Composite microbial preparation for straw treatment and preparation method

Abstract

The invention relates to the technical field of straw microbiological treatment, and discloses a composite microbial preparation for straw treatment and a preparation method. The composite microbial preparation for straw treatment is prepared by mixing a microbial group formed by pseudomonas aeruginosa, aspergillus niger, trichoderma reesei, alcaligenes faecalis and medium and low temperature pectinase with probiotics and a nutrient solution in proportion, the volume ratio of the pseudomonas aeruginosa to the aspergillus niger to the trichoderma reesei to the alcaligenes faecalis to the medium and low temperature pectinase is (1-1.5): (1-2): (0.5-1): (1.5-2): (0.5-2.5), the probiotics account for 2%-6% of the microbial group, and the mass ratio of the probiotics to the nutrient solution is 1: (20-30). The microbial preparation disclosed by the invention can well adapt to decomposition treatment of straws in cold regions, induces enrichment of aerobic bacteria, improves soil conditions, is more beneficial to survival and reproduction of the composite microbial preparation, and optimizes the decomposition treatment effect on the straws.

Description

Technical Field

[0001] The invention relates to the technical field of microbial treatment of straw, in particular to a composite microbial preparation for straw treatment and a preparation method of the microbial preparation. Background Art

[0002] Straw is a crop byproduct produced in large quantities during agricultural production. It is rich in organic matter, nitrogen (N), phosphorus (P), potassium (K) and various trace elements, and has extremely high resource utilization value. However, improper straw handling, especially open-air burning, will not only cause air pollution and increase carbon emissions, but also lead to waste of soil nutrients and structural degradation, bringing tremendous pressure to agricultural production and the ecological environment. Exploring efficient and environmentally friendly ways to utilize straw resources has become an important issue in current agricultural development and environmental protection.

[0003] At present, the mainstream method of straw treatment is centralized composting. Through composting technology, high temperature conditions and the action of microorganisms are used to decompose complex organic matter such as cellulose, hemicellulose and lignin in straw into easily absorbable nutrients and convert them into high-quality organic fertilizers. However, composting treatment requires the centralized collection of straw scattered in the field, and occupies a large area, which is often difficult to achieve in actual production, especially in the case of large-scale planting. The high cost of collection and transportation has seriously limited the promotion and application of composting technology.

[0004] Based on the above problems, in-situ treatment has gradually become a new idea for straw treatment. In-situ treatment refers to directly applying microbial agents to straw in the field, allowing the straw to decompose naturally in situ and convert it into organic matter to return to the field, without the need for centralized collection and composting. This method is easy to operate and low-cost, and is more suitable for large-scale straw treatment needs. However, in-situ treatment places higher requirements on the function and adaptability of the agent: the agent must not only be highly efficient in composting, but also be able to effectively decompose straw in the low-temperature environment of the field.

[0005] In response to the above needs, we have developed a composite microbial agent that has both composting and field in-situ treatment functions. Through this composite microbial agent, both field and composting modes can be used to meet the needs of straw treatment in different scenarios. In composting treatment, the agent can quickly and efficiently decompose the straw; in field in-situ treatment, the agent adapts to the low temperature environment, allowing the straw to decompose naturally in the field, adding organic matter to the soil and improving its structure, while reducing manual operation and transportation costs. This dual-purpose microbial agent provides a more flexible and efficient solution for the resource utilization of straw, significantly alleviating the environmental pressure of straw treatment. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a composite microbial preparation for straw treatment and a preparation method thereof, which can well adapt to the composting treatment of straw in cold regions and optimize the composting effect on the straw.

[0007] A composite microbial preparation for straw treatment comprises the following components: Pseudomonas aeruginosa (Pseudomonas aeruginosa, preservation number CGMCC.NO.26195), Aspergillus niger, Trichoderma reesei, Alcaligenes faecalis, medium-low temperature pectinase, probiotics and nutrient solution are mixed in proportion to obtain the composite microbial preparation; wherein the volume ratio (in liquid form) of Pseudomonas aeruginosa (Pseudomonas aeruginosa, preservation number CGMCC.NO.26195), Aspergillus niger, Trichoderma reesei, Alcaligenes faecalis and medium-low temperature pectinase is 1-1.5:1-2:0.5-1:1.5-2:0.5-2.5.

[0008] As a further improvement of the above scheme, the Pseudomonas aeruginosa (Pseudomonas aeruginosa, preservation number CGMCC.NO.26195), Aspergillus niger, Trichoderma reesei, Alcaligenes faecalis and mesophilic pectinase form a core microbial flora, and probiotics account for 2% to 6% of the microbial flora; the mass ratio of the probiotics to the nutrient solution is 1:20 to 30.

[0009] As a further improvement of the above scheme, in the cultivation process of Bacillus amyloliquefaciens for producing medium- and low-temperature pectinase, the composition of the liquid LB culture medium is as follows: 10 g / L NaCl, 5 g / L yeast powder, 10 g / L peptone, 15 g / L agar, and a KPB buffer with a pH of 9.0; the composition of the liquid fermentation medium A is as follows: 0.2 g / L yeast powder, 1 g / L glucose, 2 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium chloride, 0.01 g / L ferrous sulfate, 0.5 g / L magnesium sulfate, and a KPB buffer with a pH of 9.0. The invention discloses a strain of Bacillus amyloliquefaciens P17, whose deposit number is CGMCC No. 2640 and which was deposited in the General Microbiological Center of China Microorganism Culture Collection Committee on August 26, 2008. The Bacillus amyloliquefaciens is separated and purified to obtain the medium-low temperature pectinase, and the purified low temperature pectinase has the characteristic of high activity even in a low temperature environment, thus overcoming the problem that the suitable propagation temperature of conventional strains during decomposition is 28-37 DEG C, while the straw processing is mainly in winter with low environmental temperature, resulting in the limitation of the decomposition processing of straw in cold regions.

[0010] As a further improvement of the above scheme, Pseudomonas aeruginosa aeruginosa, accession number CGMCC.NO.26195) and Aspergillus niger, Trichoderma reesei, and Alcaligenes faecalis have a special liquid fermentation medium B composed of 20 g / L sodium carboxymethyl cellulose, 2 g / L dipotassium hydrogen phosphate, 1.4 g / L ammonium sulfate, 0.6 g / L magnesium sulfate heptahydrate, 0.3 g / L calcium chloride, trace amounts of ferrous sulfate, manganese sulfate, zinc chloride, and cobalt chloride; the pH is 5.5-7.5, and the culture temperature is optimally 35-40°C; the liquid fermentation medium C of Bacillus subtilis, Bacillus megaterium, and Bacillus licheniformis among the probiotics has a composition of 0.2 g / L yeast powder, 1 g / L glucose, 2 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium chloride, 0.01 g / L ferrous sulfate, 0.5 g / L magnesium sulfate, and KPB buffer with a pH of 9.0.

[0011] As a further improvement of the above scheme, the probiotics include Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis; wherein, Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis are mixed in a ratio of 0.5-1.5:0.2-0.6:0.5-1 to obtain the probiotics. In the present invention, the added Bacillus subtilis has an inhibitory and antibiotic effect on soil pathogens. In addition, it can also increase the stress resistance and nitrogen fixation of crops, thereby overcoming the phenomenon of grabbing nitrogen for crops due to the large-scale reproduction of microorganisms, resulting in the problem of yellowing and slow growth of crops. Bacillus subtilis multiplies and colonizes rapidly and in large quantities in the soil, protects plants from infection by pathogens, and secretes active substances with bactericidal effects, achieving the effect of effectively repelling, inhibiting and killing pathogens. The added Bacillus megaterium degrades organic phosphorus in the soil and converts it into harmless substances, thereby improving soil conditions to facilitate the survival and reproduction of composite microbial preparations and optimizing the composting treatment effect on straw. The added Bacillus licheniformis, as an aerobic bacterium with disease resistance and the ability to kill harmful bacteria, plays a role in increasing soil nutrients, improving soil structure, increasing the utilization rate of fertilizers, promoting the decomposition of organic matter in the soil into humus, stimulating crop growth, and fixing nitrogen, and releasing phosphorus and potassium.

[0012] As a further improvement of the above scheme, the nutrient solution is provided with a carbon source, a nitrogen source, vitamins, inorganic salts, amino acids, purines and pyrimidines. The carbon source is glucose or water-soluble chitosan, and the energy required in the microbial metabolism process is provided based on the carbon source. The nitrogen source is urea or peptone, and the protein required for the growth and reproduction of microorganisms is synthesized based on the nitrogen source. The vitamins include vitamin A, vitamin B and vitamin C, and the vitamins are used to participate in the metabolic pathways and enzymatic reactions of microorganisms. In the present invention, amino acids, purines and pyrimidines are used as growth factors to promote the production of microorganisms. The nutrient solution is composed of 20 g / L glucose or 2 g / L water-soluble chitosan, 1.5 g / L urea or 10 g / L peptone, 0.1 mg / L vitamin A, 1 mg / L vitamin B (complex of B1, B2 and B6), 0.5 mg / L vitamin C, 1.0 g / L magnesium sulfate, 1.0 g / L potassium dihydrogen phosphate, 0.5 g / L sodium chloride, 0.05 g / L iron sulfate, 0.5 g / L glutamic acid and 0.2 g / L glycine or a mixed amino acid solution (total concentration 0.2 g / L) to replace both, 0.05 g / L adenine, 0.05 g / L thymine, NaOH or HCl is used to adjust the pH to 5.5-6.5, then high-pressure sterilization is performed at 121°C for 15 minutes, and the nutrient solution is cooled for standby use, and is suitable for microorganism or cell culture.

[0013] A method for preparing a composite microbial preparation comprises the following steps:

[0014] S1 Pseudomonas aeruginosa (Pseudomonas aeruginosa, accession number

[0015] CGMCC.NO.26195) and commercially available Aspergillus niger, Trichoderma reesei, and Alcaligenes faecalis were cultured to a concentration of 5-8×10 9 CFU / ml of bacterial solution, and then 3-6×10 2 U / ml of medium-low temperature pectinase was uniformly mixed to obtain a core microbial bacterial group;

[0016] S2 cultured Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis to a concentration of 4-6×10 10 cuf / ml of bacterial solution;

[0017] S3 carbon source is the main energy source for microbial metabolism; nitrogen source supports the synthesis of microbial proteins and nucleic acids; vitamins include vitamin A, vitamin B (complex of B1, B2, B6) and vitamin C, which provide cofactors required for metabolism; inorganic salts include dipotassium hydrogen phosphate, potassium chloride, magnesium sulfate and ferrous sulfate to adjust pH and supplement mineral elements; amino acids such as glutamic acid and glycine or mixed amino acid solutions replace both to promote microbial growth; purine and pyrimidine are used as growth factors, and adenine and thymine are added respectively. When preparing, add the above ingredients in order to an appropriate amount of distilled water, stir and dissolve, adjust the total volume to 1L, and adjust the pH to 5.5-6.5 with NaOH or HCl, then sterilize at 121°C for 15 minutes, cool and set aside;

[0018] S4 mixes the probiotics and the nutrient solution;

[0019] S5: adding the microbial group prepared in S1 into the solution obtained in S4, stirring and mixing to obtain a microbial preparation.

[0020] As a further improvement of the above scheme, the preparation method is used to prepare the composite microbial preparation.

[0021] Compared with the prior art, the beneficial effects and principles of the present invention are:

[0022] The low-temperature pectinase of the present invention can efficiently decompose complex polysaccharide substances such as pectin in straw in a low-temperature (0-25°C) environment, overcoming the problem that the suitable propagation temperature of conventional strains during decomposition is 28-37°C, while straw processing is concentrated in winter with low ambient temperature, resulting in limited decomposition processing of straw in cold regions.

[0023] The composite microbial preparation of the present invention comprises a composite microbial system formed by Pseudomonas aeruginosa (Pseudomonas aeruginosa, collection number CGMCC.NO.26195) + Aspergillus niger + Trichoderma reesei + Alcaligenes faecalis, and utilizes the decomposition action of microorganisms to decompose straw, increase the organic matter content, release the nutrient elements required for plant growth, thereby increasing soil organic matter and improving soil structure.

[0024] In addition, by adding probiotics to optimize the microbial community, the enrichment of Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis was induced, and the improved soil conditions were more conducive to the survival and reproduction of the composite microbial preparations, thereby optimizing the composting treatment effect on straw.

[0025] In addition, the composite microbial agent further optimizes the straw treatment effect through synergistic effects:

[0026] 1. Principle of microbial synergy

[0027] The wall-breaking pioneer role of medium-low temperature pectinase: Medium-low temperature pectinase plays a key "pioneer" role in the entire straw degradation system. The middle gelatin layer and part of the primary cell wall of the straw are mainly composed of pectin substances. This structure is like a solid "outer line of defense", which seriously hinders the contact and degradation of microorganisms to the internal components of the straw. Medium-low temperature pectinase can specifically recognize and bind to the α-1,4-glycosidic bonds in the pectin molecules, and gradually break down the long pectin chains into small molecular fragments through hydrolysis. This hydrolysis destroys the structural integrity of the middle gelatin layer and the primary cell wall, and the connection between cells becomes loose, opening up a channel for the subsequent entry of microorganisms and enzymes.

[0028] Cellulose skeleton-destroying effect of preserved bacteria (Pseudomonas aeruginosa, collection number CGMCC.NO.26195): Pseudomonas aeruginosa (collection number CGMCC.NO.26195) has the ability to secrete specific cellulases, and the cellulase system it produces can accurately act on the cellulose skeleton structure in the primary cell wall. Cellulose exists in the primary cell wall in the form of highly ordered microfibrils, which constitute the main supporting structure of the cell wall. The endocellulase secreted by the preserved bacteria can randomly cut the β-1,4-glycosidic bonds inside the cellulose molecular chain, breaking down long-chain cellulose into shorter oligosaccharide fragments, thereby disrupting the originally tight and orderly structure of cellulose. With the destruction of the cellulose skeleton, the overall structure of the primary cell wall becomes unstable, not only releasing long cellulose chains of various forms, but also exposing the hemicellulose and lignin originally wrapped in the cell wall, providing more targets and reaction sites for the subsequent actions of other microorganisms, further promoting the process of straw degradation.

[0029] Aspergillus niger and Trichoderma reesei's comprehensive lignocellulose degradation synergistic effect: Aspergillus niger and Trichoderma reesei form an efficient "attack combination" for lignocellulose degradation. They have a rich and diverse enzyme system, including a variety of cellulases, hemicellulases and some ligninases. After the preserved bacteria break the defense line of the primary cell wall, Aspergillus niger and Trichoderma reesei can quickly colonize and penetrate into the interior. The cellulase systems of Aspergillus niger and Trichoderma reesei are complementary to those of Pseudomonas aeruginosa (preservation number CGMCC.NO.26195) in terms of action sites and enzyme activity, and can further attack the cellulose parts that are not fully decomposed in the latter, thereby improving the efficiency of cellulose degradation.

[0030] For hemicellulose, the hemicellulases secreted by Aspergillus niger and Trichoderma reesei can specifically act on various glycosidic bonds in hemicellulose, breaking it down into various monosaccharides such as xylose and arabinose, enriching the carbon source available to microorganisms. In terms of lignin degradation, although the lignin degradation capabilities of Aspergillus niger and Trichoderma reesei are relatively limited, they secrete some oxidases such as laccase and manganese peroxidase during their growth. These enzymes can participate in the oxidative degradation process of lignin, degrading it by gradually breaking down the complex three-dimensional network structure of lignin. The synergistic effect of the two achieves extensive and in-depth degradation of lignocellulose in straw, releasing a large amount of sugars and other nutrients, providing sufficient carbon sources and energy for the growth and metabolism of the entire microbial community.

[0031] The nitrogen guarantee role of Alcaligenes faecalis: The nitrogen content in straw is relatively low, which is difficult to meet the nitrogen demand of microorganisms during their large-scale growth and reproduction. Alcaligenes faecalis plays a vital role in nitrogen conversion and supply in this process. It has a unique nitrogenase system that can convert nitrogen in the air and nitrogen nutrients in the environment into ammonia nitrogen or nitrate nitrogen that can be used by microorganisms at normal temperature and pressure. This nitrogen fixation process involves complex electron transfer and energy metabolism. The molybdenum iron protein in the nitrogenase serves as the active center. With the cooperation of iron protein providing electrons and energy, nitrogen molecules are gradually reduced to ammonia. Part of the generated ammonia nitrogen is assimilated by Alcaligenes faecalis itself for cell synthesis, and the other part quickly diffuses into the surrounding environment, providing a key nitrogen source for microorganisms such as Pseudomonas aeruginosa, Aspergillus niger, and Trichoderma reesei, maintaining the stable growth and metabolic activity of the microbial flora, and ensuring that the straw degradation process can proceed continuously and efficiently.

[0032] 2. Soil ecological optimization and nutrient activation effects of probiotics:

[0033] The probiotics composed of Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis play multiple roles in the straw degradation process. In the later stage of straw degradation, as the straw gradually decomposes into soil organic matter, these probiotics can colonize in the soil and have a positive impact on the soil microecological environment; the probiotics will secrete some extracellular enzymes and small molecular metabolites during the growth and metabolism process. These substances can participate in the transformation and activation of nutrients in the soil, interact with other microorganisms in the soil, form a complex microbial community network, promote the circulation and transformation of various nutrients in the soil, improve the effectiveness of soil nutrients, and provide more sufficient nutrient supply for crop growth.

[0034] 3. Early nutritional support and microbial adaptation promotion of nutrient solution: Nutrient solution plays a vital role in nutritional support in the early stage of the compound microbial preparation. Adequate supply of carbon and nitrogen sources avoids the reduction of microbial activity due to nutrient deficiency, ensuring that the straw degradation process can be started quickly and efficiently in the initial stage. At the same time, other nutrients such as vitamins help the microorganisms in the agent to better adapt to the environment, promote their proliferation on the straw surface and the surrounding environment, and enhance the overall vitality and degradation ability of the microbial community. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a bar graph showing the change of straw dry matter degradation rate in the soil of treatment groups 1-9 over time in comparative example 1 of the present invention under a field environment of 8-15°C.

[0036] Figure 2 It is a bar graph showing the change of cellulose degradation rate in soil of treatment groups 1-9 over time in comparative example 1 of the present invention under a field environment of 8-15°C.

[0037] Figure 3 It is a bar graph showing the change of hemicellulose degradation rate in the soil of treatment groups 1-9 over time in comparative example 1 of the present invention under a field environment of 8-15°C.

[0038] Figure 4 It is a bar graph showing the change of the degradation rate of lignocellulose in the soil of treatment groups 1-9 over time in comparative example 1 of the present invention under a field environment of 8-15°C. DETAILED DESCRIPTION

[0039] The specific embodiments of the present invention are described in detail below.

[0040] Example 1

[0041] This embodiment provides a composite microbial preparation for straw treatment, which has the following components: Pseudomonas aeruginosa (Pseudomonas aeruginosa, accession number CGMCC.NO.26195), Aspergillus niger, Trichoderma reesei, Alcaligenes faecalis, medium-low temperature pectinase, probiotics and nutrient solution are mixed in proportion to obtain the composite microbial preparation. Among them, the indicators of Pseudomonas aeruginosa (Pseudomonas aeruginosa, accession number CGMCC.NO.26195), Aspergillus niger, Trichoderma reesei, Alcaligenes faecalis, medium-low temperature pectinase are respectively

[0042] 5.53×10 9 cuf / ml,5.84×10 9 cuf / ml,7.77×10 9 cuf / ml,6.75×10 9cuf / ml and 4.45×10 2 U / ml, the volume ratio is 1:1:0.5:1.5:0.5.

[0043] The Pseudomonas aeruginosa described in this application was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on December 14, 2022, with the deposit number: CGMCC No.26195.

[0044] The probiotics include Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis. The indexes of Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis are 5.55×10 10 cuf / ml, 4.48×10 10 cuf / ml, 4.12×10 10 cuf / ml, and mixed in a ratio of 0.5:0.2:0.5 to obtain the probiotics.

[0045] Nutrient solution composition: glucose 20g / L, urea 1.5g / L, vitamin A 0.1mg / L, vitamin B (complex of B1, B2, B6) 1mg / L, vitamin C 0.5mg / L, magnesium sulfate 1.0g / L, potassium dihydrogen phosphate 1.0g / L, sodium chloride 0.5g / L, iron sulfate 0.05g / L, mixed amino acid solution 0.2g / L, adenine 0.05g / L, thymine 0.05g / L, pH to 5.8, complete high-pressure sterilization at 121°C for 15 minutes and cooling.

[0046] The Pseudomonas aeruginosa (Pseudomonas aeruginosa, accession number

[0047] CGMCC.NO.26195), Aspergillus niger, Trichoderma reesei, Alcaligenes faecalis and mesophilic pectinase form a microbial flora, and probiotics account for 2% of the microbial flora, and the mass ratio of the probiotics to the nutrient solution is 1:20.

[0048] The preparation method of the composite microbial preparation of this embodiment comprises the following steps:

[0049] S1 Pseudomonas aeruginosa (Pseudomonas aeruginosa, accession number

[0050] CGMCC.NO.26195), Aspergillus niger, Trichoderma reesei, and Alcaligenes faecalis were cultured to a concentration of 5-8×10 9 cfu / ml of bacterial solution, and then mixed with medium-low temperature pectinase at a volume ratio of 1:1:0.5:1.5:0.5 to obtain a microbial bacterial group;

[0051] S2 takes probiotics accounting for 2% of the microbial group, and mixes the probiotics and nutrient solution in a ratio of 1:20;

[0052] S3: adding the microbial flora into the solution obtained in S2, stirring and mixing to obtain a microbial preparation.

[0053] Example 2

[0054] This embodiment provides a composite microbial preparation for straw treatment, which has the following components: Pseudomonas aeruginosa (Pseudomonas aeruginosa, accession number CGMCC.NO.26195), Aspergillus niger, Trichoderma reesei, Alcaligenes faecalis, medium-low temperature pectinase, probiotics and nutrient solution are mixed in proportion to obtain the composite microbial preparation. Among them, the indicators of Pseudomonas aeruginosa (Pseudomonas aeruginosa, accession number CGMCC.NO.26195), Aspergillus niger, Trichoderma reesei, Alcaligenes faecalis, medium-low temperature pectinase are respectively

[0055] 5.88×10 9 cuf / ml,7.31×10 9 cuf / ml,6.33×10 9 cuf / ml,7.28×10 9 cuf / ml and 5.11×10 2 U / ml, volume ratio is 1.5:2:1:2:2.5.

[0056] The probiotics include Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis. The indexes of Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis are 4.25×10 10 cuf / ml, 5.54×10 10 cuf / ml, 5.78×10 10 cuf / ml, and mixed in a ratio of 1.5:0.6:1 to obtain the probiotics.

[0057] Nutrient solution composition: glucose 20g / L, urea 1.5g / L, vitamin A 0.1mg / L, vitamin B (complex of B1, B2, B6) 1mg / L, vitamin C 0.5mg / L, magnesium sulfate 1.0g / L, potassium dihydrogen phosphate 1.0g / L, sodium chloride 0.5g / L, iron sulfate 0.05g / L, mixed amino acid solution 0.2g / L, adenine 0.05g / L, thymine 0.05g / L, pH to 5.8, complete high-pressure sterilization at 121°C for 15 minutes and cooling.

[0058] The Pseudomonas aeruginosa (Pseudomonas aeruginosa, accession number

[0059] CGMCC.NO.26195), Aspergillus niger, Trichoderma reesei, Alcaligenes faecalis and mesophilic pectinase form a microbial flora, and probiotics account for 6% of the microbial flora, and the mass ratio of the probiotics to the nutrient solution is 1:30.

[0060] The preparation method of the composite microbial preparation of this embodiment comprises the following steps:

[0061] S1: Pseudomonas aeruginosa (CGMCC.NO.26195), Aspergillus niger, Trichoderma reesei, and Alcaligenes faecalis were cultured to a concentration of 5-8×10 9 cfu / ml of bacterial solution, and then mixed with medium-low temperature pectinase in a volume ratio of 1.5:2:1:2:2.5 to obtain a microbial bacterial group;

[0062] S2 takes probiotics accounting for 6% of the microbial group and mixes the probiotics and nutrient solution at a ratio of 1:30;

[0063] S3: adding the microbial flora into the solution obtained in S2, stirring and mixing to obtain a microbial preparation.

[0064] The above embodiments are merely preferred implementations of the present invention. Any simple modification, amendment and substitution of the above embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

[0065] Comparative Example 1

[0066] Two commercially available microbial agents were used as the control group and the composite microbial agent of Example 1 was used for the test. The specific test process was as follows: The long-term "wheat-rice-rice" grain three-crop rotation planting area in Zhejiang was selected, and the test plot was divided into 4m×4m plots. Each treatment was repeated 3 times and arranged in random blocks. A 0.5-1m isolation zone was left between treatments in each test plot to prevent the spread of the agent. Before the experiment, the straw residue was cleaned uniformly to ensure consistent soil conditions.

[0067] Set up field trial treatments:

[0068] Treatment 1: natural decomposition of straw (control group, no bacterial agent added);

[0069] Treatment 2: the composite microbial agent of Example 1 (applied at the recommended dose, 3 kg / mu);

[0070] Treatment 3: the composite microbial agent of Example 1, only without the medium-low temperature pectinase (applied at the recommended dosage, 3 kg / mu);

[0071] Treatment 4: the composite microbial agent of Example 1, except that it does not contain Pseudomonas aeruginosa (Pseudomonas aeruginosa, accession number CGMCC.NO.26195) (applied at the recommended dosage, 3 kg / mu);

[0072] Treatment 5: the composite microbial agent of Example 1, except that Aspergillus niger and Trichoderma reesei were not included (applied at the recommended dosage, 3 kg / mu);

[0073] Treatment 6: the composite microbial agent of Example 1, except that Alcaligenes faecalis was not included (applied at the recommended dosage, 3 kg / mu);

[0074] Treatment 7: the composite microbial agent of Example 1, but without the probiotic combination (administered at the recommended dosage, 3 kg / mu);

[0075] Treatment 8: Shandong Sapphire straw treatment agent (applied according to the manufacturer's recommended dosage, 5 kg / mu);

[0076] Treatment 9: Guangdong Weiliwang straw treatment bacteria agent (applied according to the manufacturer's recommended dosage, 2kg / mu).

[0077] When the harvester harvests rice, the straw is crushed synchronously and evenly covered on the deep pits or open spaces in the fields. In the field environment of 8-15°C, randomly take the ground straw (water content is 40%-50%), and use each microbial agent treatment group to dissolve the microbial agent in water appropriately and spray it evenly on the straw, so that the humidity of the straw material is 60%-80%, which is conducive to the microbial growth of the agent and the decomposition of the straw. The treated straw is packed into nylon mesh bags (pore size 1-2mm) at 100g per bag, and the initial weight is recorded. It is manually buried in the 5cm-10cm tillage layer of each corresponding test plot (3*9=27 repetitions) to simulate the operating depth of the field rotary tiller, and the nylon bags are taken out after 30, 60, and 90 days to measure the parameters.

[0078] analyze Figure 1-4 It can be seen that: under the field environment of 8-15℃, the degradation of straw dry matter and various lignocelluloses in treatment group 1 (control group) was the worst, while the degradation of straw dry matter and various lignocelluloses in treatment group 2 (composite microbial agent) was the best. The bacteria in treatment groups 3, 4, 5, 6, and 7 were better than those in treatment group 1 (control group) in all aspects, and worse than those in treatment group 2, which also proves the importance and functional specificity of each component in the agent. Treatment group 8 (Shandong Sapphire Straw Treatment Agent) was inferior to treatment group 2 in all aspects; treatment group 9 (Guangdong Weiliwang Straw Treatment Agent) was slightly better than treatment group 2 in lignin degradation rate, but poor in other aspects. The above results prove that the composite microbial agent of the present invention can adapt well to the composting treatment of straw in cold regions, and has excellent straw composting effect.

[0079] Comparative Example 2:

[0080] The same as comparative example 1, two commercially available microbial agents were used as the control group and the composite microbial agent of Example 1 was tested. The specific test process was as follows: the wheat planting area of ​​Weifang, Shandong was selected, and the test plot was divided into 8m×8m plots. Each treatment was repeated 3 times and arranged in random blocks. A 0.5-1m isolation zone was left between treatments in each test plot to prevent the spread of the agent. Before the experiment, the straw residue was cleaned uniformly to ensure consistent soil conditions.

[0081] The field test treatments were set up, the same as in comparative example 1, with a total of treatment groups 1-9.

[0082] When the harvester harvests wheat, the straw is crushed synchronously and evenly covered in the deep pits or open spaces in the fields. In the field environment of 0-6°C, randomly take the ground straw (water content is 35%-55%), and use each microbial agent treatment group to dissolve the microbial agent in water appropriately and spray it evenly on the straw, so that the humidity of the straw material is 60%-80%, which is conducive to the microbial growth of the agent and the decomposition of the straw. The treated straw is artificially buried in the 5cm-10cm of the tillage layer of each corresponding test plot (3*9=27 repetitions) according to the proportion of natural coverage, simulating the operating depth of the field rotary tiller, and taking soil samples from each plot on the 0th day and the 90th day to measure the parameters. The results of the determination of cellulase, cellulase, polyphenol oxidase, peroxidase, organic matter, alkaline nitrogen, and soil microbial biomass carbon MBC in the soil of treatment groups 1-9 are shown in Table 1.

[0083] Table 1 Test results of soil in treatment groups 1-9

[0084]

[0085]

[0086] From the analysis of Table 1, it can be seen that after 90 days of treatment under the field environment of 0-6℃, there was no significant change in the cellulase, polyphenol oxidase and organic matter of treatment group 1 (control group), while xylanase, peroxidase, alkaline nitrogen and soil microbial biomass carbon MBC were significantly improved; all indicators of other treatment groups were significantly improved. All indicators of treatment group 2 (composite microbial agent) were not only better than those of treatment group 1, but also the best or one of the best among all other combinations, so treatment group 2 was the combination with the best comprehensive ability; there was no significant difference in polyphenol oxidase and peroxidase between treatment group 2 and treatment group 8 (Shandong Sapphire Straw Treatment Agent) and treatment 9 (Guangdong Weiliwang Straw Treatment Agent), which also corresponds to the phenomenon that the three have similar lignin degradation abilities in comparative example 1. At the same time, the soil microbial biomass carbon MBC of treatment 7 was 303.11±1.32e, which was only higher than that of treatment group 1 but lower than that of all other treatment groups, which also illustrates the importance of probiotics (Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis) in activating the soil microecological environment.

[0087] Comparative Example 3

[0088] Two commercially available microbial agents were used as the control group and the composite microbial agent of Example 2 was tested. The specific test process was as follows: The long-term "wheat-rice-rice" grain three-crop rotation planting area in Zhejiang was selected, and the test plot was divided into 5m×5m plots. Each treatment was repeated 3 times and arranged in random blocks. A 0.5-1m isolation zone was left between treatments in each test plot to prevent the spread of the agent. Before the experiment, the straw residue was cleaned uniformly to ensure consistent soil conditions.

[0089] Set up field trial treatments:

[0090] Treatment 1: natural decomposition of straw (control group, no bacterial agent added);

[0091] Treatment 2: the composite microbial agent of Example 1 (applied at the recommended dose, 3 kg / mu);

[0092] Treatment 3: the composite microbial agent of Example 1, only without the medium-low temperature pectinase (applied at the recommended dosage, 3 kg / mu);

[0093] Treatment 4: the composite microbial agent of Example 1, except that it does not contain Pseudomonas aeruginosa (Pseudomonas aeruginosa, accession number CGMCC.NO.26195) (applied at the recommended dosage, 3 kg / mu);

[0094] Treatment 5: the composite microbial agent of Example 1, but without the probiotic combination (administered at the recommended dosage, 3 kg / mu);

[0095] Treatment 6: Shandong Sapphire straw treatment agent (applied according to the manufacturer's recommended dosage, 5 kg / mu);

[0096] Treatment 7: Guangdong Weiliwang straw treatment bacteria agent (applied according to the manufacturer's recommended dosage, 2kg / mu).

[0097] When the harvester harvests winter wheat in April, the straw is crushed synchronously and evenly covered in deep pits or open spaces in the fields. In a field environment of 0-8°C, randomly take ground straw (moisture content of 40%-50%), and use each microbial agent treatment group to appropriately dissolve the microbial agent in water and spray it evenly on the straw, so that the humidity of the straw material is 60%-80%, which is conducive to the microbial growth of the agent and the decomposition of the straw. The treated straw is manually buried in the 5cm-10cm tillage layer of each corresponding test plot (3*7=21 repetitions) according to the proportion of natural coverage, simulating the operating depth of the field rotary tiller, planting early rice and harvesting in July, taking rice samples to measure relevant indicators, and the results are shown in Table 2. Table 2 Results of the number of grains per ear, number of effective ears, fruit set rate, thousand-grain weight and yield of rice samples in treatments 1-7 test plots

[0098]

[0099]

[0100] Analysis of Table 2 shows that after returning the straw to the field in situ under a field environment of 0-8℃, the yield level of early rice was measured. There was no significant difference in the number of panicles among all treatment groups. There was no significant difference in the number of effective panicles between treatment group 1 (control group) and treatment groups 3, 4, and 5, and it was lower than treatment groups 2, 6 (Shandong Sapphire straw treatment microbial agent), and 7 (Guangdong Weiliwang straw treatment microbial agent); but the fruiting rate and thousand-grain weight of treatment group 1 were significantly lower than those of other treatment groups, and treatment group 2 was the best. Similar to the number of effective panicles, there was no significant difference in yield between treatment group 1 (control group) and treatment groups 3, 4, and 5, and it was the lowest level among all treatment groups, indicating that the lack of components reduced the yield efficacy of the composite microbial agent; the yield of treatment group 2 was the highest level, and there was no significant difference between treatment groups 6 and 7 and it was the second highest level. Taking all factors into consideration, treatment group 2 can significantly promote the increase of rice yield.

[0101] Example 3

[0102] This embodiment provides the application of the composite microbial preparation of embodiment 1 or 2 in returning straw to the field. After the crops are harvested, the straw does not need to be crushed, but is directly treated in situ in the field in its natural form. The specific operation is as follows:

[0103] According to the actual situation of the straw-covered area, the composite microbial preparation is evenly spread on the ground. The dosage of the microbial preparation is: the ratio of straw to composite microbial preparation is 200-500:1 (ratio of straw mass to microbial preparation mass).

[0104] Specific operation: Spread 2-5kg compound microbial agent per ton of straw. If the straw is dense (such as a large amount of straw coverage in a single field), the amount of microbial agent can be appropriately increased to 5kg / t straw.

[0105] Coverage: 2-5 kg ​​compound fungus agent per mu of land (depending on the amount of straw covering). Thin straw covering: 2-3 kg / mu; thick straw covering: 2-5 kg / mu.

[0106] Operation method: Broadcasting method: Spread the microbial agent evenly on the surface of the straw; Shallow burial method (optional): Combine mechanical tillage to mix the microbial agent, straw and surface soil (5-10 cm) to improve the contact efficiency between the microbial agent and the straw.

[0107] Humidity management: Make full use of natural precipitation. If the weather is dry, add water appropriately to keep the humidity of the straw at about 60%. It is best to do it before it rains, as rainwater will increase the rate of decomposition. Then, turn the straw and the soil together mechanically or manually to make them fully mixed and in contact, which will promote the rapid decomposition and utilization of the straw by microorganisms.

[0108] This method not only reduces the mechanical cost required for straw crushing, but also effectively improves soil structure and increases soil organic matter content, while avoiding environmental pollution caused by straw burning. It has the advantages of simple operation and efficient use of resources.

Claims

1. A composite microbial preparation for straw treatment, characterized in that: The composite microbial preparation comprises the following components: Pseudomonas aeruginosa, Aspergillus niger, Trichoderma reesei, Alcaligenes faecalis, medium-low-temperature pectinase, probiotics and nutrient solution are mixed in proportion to obtain the composite microbial preparation; wherein the volume ratio of Pseudomonas aeruginosa, Aspergillus niger, Trichoderma reesei, Alcaligenes faecalis and medium-low-temperature pectinase is 1-1.5:1-2:0.5-1:1.5-2:0.5-2.

5.

2. The composite microbial preparation for straw treatment according to claim 1, characterized in that: The Pseudomonas aeruginosa, Aspergillus niger, Trichoderma reesei, Alcaligenes faecalis and mesophilic pectinase form a microbial flora, and the probiotics account for 2% to 6% of the microbial flora; the Pseudomonas aeruginosa is deposited with CGMCC.NO.26195; The mass ratio of the probiotics to the nutrient solution is 1:20-30.

3. The composite microbial preparation for straw treatment according to claim 1, characterized in that: The strain Bacillus amyloliquefaciens P17 has a deposit number of CGMCC No. 2640 and was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration on August 26, 2008. The medium-low temperature pectinase is obtained by separation and purification of Bacillus amyloliquefaciens; The method for obtaining the medium-low temperature pectinase by purification is specifically performed as follows: Step 1: Inoculate Bacillus amyloliquefaciens into liquid LB medium and culture with shaking at 13-25°C until the exponential growth phase; Step 2: Transfer the bacterial liquid in the exponential growth phase to liquid fermentation medium A, and culture at 13-25°C with shaking until the early stable phase, which is the peak of enzyme production. Centrifuge the fermentation liquid, take the supernatant, and filter with filter paper to obtain the crude enzyme liquid; Step 3: Use a membrane package with a molecular weight cutoff of 10KDa to concentrate the volume of the crude enzyme solution to 1 / 10 of the original volume, add ammonium sulfate to the concentrated enzyme solution to 60% to 70% saturation, centrifuge to obtain the supernatant, continue to add ammonium sulfate to 90% to 100%, centrifuge to obtain the precipitate, and dialyze in KPB buffer with a pH of 8.0 overnight; Step 4: Centrifuge the dialyzed enzyme solution, take the supernatant, load it onto a DEAE-Sepharose pre-packed column equilibrated with KPB buffer at pH 8.0, collect the active permeate peak enzyme solution, and concentrate it using a concentrator with a molecular weight cutoff of 10 KDa; Step 5: dialyze the concentrated enzyme solution overnight with KPB buffer at pH 6.0, centrifuge and take the supernatant, load it onto a CM-Sepharose pre-packed column that has been equilibrated with KPB buffer at pH 6.0, elute and collect the active part to obtain the low-temperature pectinase with an enzyme activity of 3-6×10 2 U / ml.

4. The composite microbial preparation for straw treatment according to claim 3, characterized in that: The composition of the liquid LB medium is as follows: NaCl 10 g / L, yeast powder 5 g / L, peptone 10 g / L, agar 15 g / L, KPB buffer with a pH of 9.0; The composition of the liquid fermentation medium A is as follows: 0.2 g / L yeast powder, 1 g / L glucose, 2 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium chloride, 0.01 g / L ferrous sulfate, 0.5 g / L magnesium sulfate, and KPB buffer with a pH of 9.

0.

5. The composite microbial preparation for straw treatment according to claim 1, characterized in that: Pseudomonas aeruginosa, Aspergillus niger, Trichoderma reesei, and Alcaligenes faecalis were prepared by fermentation in liquid fermentation medium B and reached 5-8×10 9 cuf / ml; The composition of the liquid fermentation culture B is as follows: 20 g / L sodium carboxymethyl cellulose, 2 g / L dipotassium hydrogen phosphate, 1.4 g / L ammonium sulfate, 0.6 g / L magnesium sulfate heptahydrate, 0.3 g / L calcium chloride, trace amounts of ferrous sulfate, manganese sulfate, zinc chloride, and cobalt chloride; the pH is 5.5-7.5, and the culture temperature is optimally 35-40°C.

6. The composite microbial preparation for straw treatment according to claim 1, characterized in that: The probiotics include Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis, and the composition of the liquid fermentation medium C is as follows: 0.2 g / L yeast powder, 1 g / L glucose, 2 g / L ammonium sulfate, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium chloride, 0.01 g / L ferrous sulfate, 0.5 g / L magnesium sulfate, and KPB buffer with a pH of 9.

0.

7. The composite microbial preparation for straw treatment according to claim 1, characterized in that: The probiotics include Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis; Among them, Bacillus subtilis, Bacillus megaterium and Bacillus licheniformis were prepared by fermentation in liquid fermentation medium C and reached 4-6×10 10 cuf / ml, and mixed in a ratio of 0.5-1.5:0.2-0.6:0.5-1 to obtain the probiotics.

8. The composite microbial preparation for straw treatment according to claim 1, characterized in that: The nutrient solution contains a carbon source, a nitrogen source, vitamins, inorganic salts, amino acids, purines and pyrimidines; The carbon source is glucose or water-soluble chitosan, the nitrogen source is urea or peptone, and the vitamins include vitamin A, vitamin B and vitamin C, and the composition is as follows: Glucose 20g / L or water-soluble chitosan 2g / L, urea 1.5g / L or peptone 10g / L, vitamin A 0.1mg / L, vitamin B1mg / L, vitamin C 0.5mg / L, magnesium sulfate 1.0g / L, potassium dihydrogen phosphate 1.0g / L, sodium chloride 0.5g / L, ferrous sulfate 0.05g / L, glutamic acid 0.5g / L and glycine 0.2g / L or mixed amino acid solution (total concentration 0.2g / L) to replace both, adenine 0.05g / L, thymine 0.05g / L, use NaOH or HCl to adjust the pH to 5.5-6.5, then sterilize at 121℃ for 15min, cool and set aside, suitable for microbial or cell culture.

9. A method for preparing a composite microbial preparation, characterized in that: The steps include: S1: Pseudomonas aeruginosa, Aspergillus niger, Trichoderma reesei, and Alcaligenes faecalis were cultured to a concentration of 5-8×10 9 cfu / ml of bacterial solution, and then mixed with medium-low temperature pectinase to obtain a microbial bacterial group; S2 mixes the probiotics and the nutrient solution; S3: adding the microbial flora into the solution obtained in S2, stirring and mixing to obtain the microbial preparation as claimed in any one of claims 1 to 8.

10. Use of the composite microbial preparation according to any one of claims 1 to 8 in returning straw to the field, characterized in that: The specific operation of the application is as follows: according to the actual situation of the straw coverage area, the composite microbial preparation is evenly spread on the ground, and the mass ratio of straw to composite microbial preparation is 200-500:1, or the amount of composite microbial preparation is controlled within the range of 2-5 kg / mu to ensure that the preparation can cover the surface of the straw.

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