A compound microbial agent for promoting low-temperature decomposition of rice straw, a microcapsule and a preparation method and application thereof

Abstract

The application discloses a kind of composite microbial inoculant for promoting low-temperature decomposition of rice straw, microcapsule and its preparation method and application, belong to microbial inoculant technical field.The composite microbial inoculant is composed of Sphingobium strain b362:SSTF3, Paecilomyces strain f206:SSTF1 and Flavobacterium strain BB-43, and the volume ratio of the three bacteria liquids is preferably 0.377:0.397:0.226.The microcapsule composite inoculant is formed by embedding the low-temperature composite bacterial system as active component with sodium alginate-chitosan composite wall material;Wherein, sodium alginate is crosslinked to form gel beads by Ca 2+ Chitosan is coated on the surface of the gel beads.During preparation, the mixture of sodium alginate-composite bacteria liquid containing the low-temperature composite bacterial system is dropped into calcium chloride solution to crosslink into balls, and then coated with chitosan solution to obtain the microcapsule composite inoculant.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, and in particular to a compound microbial inoculant, microcapsules, preparation method and application for promoting low-temperature decomposition of rice straw. Background Technology

[0002] Rice straw is an important agricultural biomass resource. Returning straw to the field allows organic carbon and mineral nutrients such as nitrogen, phosphorus, and potassium to return to the soil, which is of great significance for improving soil structure, enhancing topsoil fertility, and reducing air pollution caused by open burning of straw.

[0003] The main structural components of rice straw are cellulose, hemicellulose, and lignin. Hemicellulose and lignin cross-link to form a dense network, while lignin coats or connects cellulose microfibrils, giving the straw a strong natural barrier against degradation. Under low-temperature conditions, the metabolic activity and extracellular enzyme activity of native microorganisms are inhibited, resulting in slow straw decomposition, insufficient structural damage, and a long decomposition cycle. This can easily affect land preparation after returning straw to the field in autumn and winter, as well as the sowing of subsequent crops.

[0004] Inoculating with exogenous straw decomposing microbial agents is an important way to improve straw decomposition efficiency. Most existing straw decomposing agents are liquid agents or traditional solid carrier agents. Under suitable temperature conditions, they can play a certain role in promoting decomposition. However, under stress conditions such as low temperature, ultraviolet radiation, freeze-thaw cycles, and fluctuations in field moisture, the number of viable exogenous bacteria decreases rapidly, and their colonization ability and persistence are insufficient, making it difficult to continuously exert a degradation effect.

[0005] Furthermore, the degradation of lignocellulose in rice straw involves the synergistic action of multiple enzyme systems. A single bacterial strain typically struggles to simultaneously achieve efficient degradation of cellulose, hemicellulose, and lignin; simple empirically formulated compound microbial agents may suffer from strain antagonism, imbalanced ratios, or insufficient low-temperature synergy. Therefore, it is necessary to construct compound microbial systems that are highly adaptable to low temperatures, have good compatibility with other enzymes, and possess synergistic degradation capabilities across multiple enzyme systems.

[0006] Microencapsulation technology can provide a relatively stable microenvironment for live bacteria, reducing damage to the bacteria from external physical and chemical stresses, and enabling continuous release of bacteria under suitable conditions. The sodium alginate-chitosan system has mild gelation conditions, good biocompatibility, and a wide range of wall material sources, making it a promising candidate for agricultural microbial preparations. For the scenario of returning rice straw to the field at low temperatures, constructing a microencapsulated composite microbial agent that combines low-temperature adaptability, multi-enzyme synergistic degradation ability, and formulation stability has practical application value for improving the decomposition efficiency of straw in autumn and winter. Summary of the Invention

[0007] The purpose of this invention is to provide a compound microbial agent, microcapsules, preparation method and application for promoting the low-temperature decomposition of rice straw, so as to solve the problems of rapid activity decay and insufficient residual effect of exogenous degrading bacteria and low decomposition efficiency of rice straw under low temperature conditions.

[0008] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a compound microbial agent for promoting low-temperature decomposition of rice straw, comprising *Sphingomonas* (…). Sphingobacterium sp.) b362: SSTF3, Penicillium ( Paecilomyces sp.) f206: SSTF1 and Flavobacterium ( Flavobacterium sp.) BB-43; The *Sphingomonas* b362:SSTF3 was deposited on June 4, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251237. The *Paecilomyces f206:SSTF1* was deposited on March 20, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025548. The Flavobacterium BB-43 was deposited at the China Center for Type Culture Collection (CCTCC) on June 4, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20261191.

[0009] The second technical solution of the present invention is a microcapsule for promoting low-temperature decomposition of rice straw, comprising the aforementioned composite microbial agent.

[0010] The third technical solution of the present invention, the method for preparing the microcapsules, includes the following steps: (1) The composite microbial agent is mixed with sodium alginate solution to obtain sodium alginate-bacterial solution mixture; (2) The sodium alginate-bacterial solution mixture is dropped into calcium chloride solution for ionic cross-linking and solidification to obtain sodium alginate gel microspheres; (3) The sodium alginate gel microspheres are coated in chitosan solution to obtain sodium alginate-chitosan microcapsule composite bacterial agent.

[0011] The fourth technical solution of the present invention is the application of the composite microbial agent or the microcapsule in promoting the low-temperature decomposition of rice straw.

[0012] Based on the above technical solution, the present invention has the following technical effects: The low-temperature composite microbial system disclosed in this invention is composed of bacteria and fungi that grow stably at low temperatures without obvious antagonism, and can form functionally complementary enzyme systems for degrading cellulose, hemicellulose, and lignin under low-temperature conditions; the sodium alginate-chitosan composite wall material is obtained through Ca... 2+ The composite network structure formed by ionic cross-linking, hydrogen bonding and electrostatic interaction can improve the activity retention of bacteria under freeze-thaw and ultraviolet stress; the microcapsule composite bacterial agent can reduce the mechanical strength of rice straw under simulated field return conditions at 10℃, promote the reduction of residual cellulose, hemicellulose and lignin, and promote the release of nutrients such as nitrogen, phosphorus and potassium in straw. Attached Figure Description

[0013] Figure 1 This is a colony morphology diagram of the candidate strains. A represents *Sphingomonas* (…). Sphingobacterium sp.) b362: Colony morphology of SSTF3, B represents Penicillium pseudocarpa (sp.) Paecilomyces sp.) f206: SSTF1 colony morphology, C is Flavobacterium ( Flavobacterium Colony morphology of sp. BB-43.

[0014] Figure 2 The values ​​represent the enzyme activities of the candidate strains. A represents cellulase activity, B represents filter paper enzyme activity, and C represents laccase activity.

[0015] Figure 3 The effect of different candidate composite bacterial strains on the weight loss rate of rice straw at 10℃.

[0016] Figure 4 This is a schematic diagram of the preparation process of sodium alginate-chitosan microcapsule composite bacterial agent.

[0017] Figure 5 The effects of different process parameters on the microcapsule encapsulation efficiency are shown. A represents the effect of sodium alginate concentration on encapsulation efficiency, B represents the effect of chitosan concentration on encapsulation efficiency, C represents the effect of calcium chloride concentration on encapsulation efficiency, and D represents the effect of time on encapsulation efficiency.

[0018] Figure 6 The image shows the appearance and scanning electron microscope (SEM) image of the microcapsule spheres.

[0019] Figure 7 The image shows the Fourier transform infrared spectrum of the microcapsule compound bacterial agent.

[0020] Figure 8 The differences in freeze-thaw resistance, UV resistance, and acid-base resistance between microencapsulated compound bacterial agents and free compound bacterial solutions are shown. A represents the difference in freeze-thaw resistance, B represents the difference in acid-base resistance, and C represents the difference in UV resistance.

[0021] Figure 9 A comparison chart of the mechanical strength of rice straw under different treatments.

[0022] Figure 10 Figure 1 shows the contents of cellulose (A), hemicellulose (B), and lignin (C) in rice straw under different treatments after 60 days of low-temperature degradation.

[0023] Figure 11 Scanning electron microscope images of rice straw under different treatments after 60 days of low-temperature degradation.

[0024] Figure 12 Fourier transform infrared spectra of rice straw under different treatments after 60 days of low-temperature degradation.

[0025] Figure 13 X-ray diffraction crystallinity diagrams of rice straw under different treatments after 60 days of low-temperature degradation.

[0026] Figure 14 Figure 1 shows the residual nitrogen (A), phosphorus (B), and potassium (C) content of rice straw under different treatments after 60 days of low-temperature degradation. Detailed Implementation

[0027] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0028] Unless otherwise stated, all percentages mentioned herein are mass-volume percentages; the low temperature refers to 5–15°C, preferably 10°C. The numerical ranges given in this invention should be understood as disclosing various intermediate values ​​between the upper and lower limits of the range, as well as smaller ranges composed of any upper and lower limit values.

[0029] The "bacterial liquid" mentioned in this invention includes bacterial fermentation broth, fungal spore suspension, fungal hyphae suspension, or a mixture thereof; for f206:SSTF1, it can be balanced by the number of culturable units, the number of spores, or a conversion method corresponding to the number of culturable units.

[0030] This invention provides a compound microbial agent for promoting the low-temperature decomposition of rice straw, including *Sphingomonas* (…). Sphingobacterium sp.) b362: SSTF3, Penicillium ( Paecilomyces sp.) f206: SSTF1 and Flavobacterium ( Flavobacterium sp.) BB-43; The *Sphingomonas* b362:SSTF3 was deposited on June 4, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251237. The *Paecilomyces f206:SSTF1* was deposited on March 20, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025548. The Flavobacterium BB-43 was deposited at the China Center for Type Culture Collection (CCTCC) on June 4, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20261191.

[0031] In some specific implementations, the volume ratio of the bacterial culture of *Sphingosporobacter b362:SSTF3*, *Penicillium f206:SSTF1*, and *Flavobacterium BB-43* is 0.377:0.397:0.226. The culture volume of *Sphingomonas b362:SSTF3*, *Penicillium f206:SSTF1*, and *Flavobacterium BB-43* is 1 × 10⁻⁶ units. 8 ~1×10 9 CFU / mL.

[0032] This invention also provides a microcapsule for promoting low-temperature decomposition of rice straw, comprising the aforementioned composite microbial agent and a sodium alginate-chitosan composite wall material encapsulating the low-temperature composite microbial system.

[0033] In some specific implementations, the wall material of the microcapsules includes sodium alginate, calcium chloride, and chitosan.

[0034] Sodium alginate-chitosan composite wall material includes Ca 2+ Cross-linked sodium alginate gel microspheres and a chitosan layer coating the surface of the sodium alginate gel microspheres.

[0035] The microcapsules have a wet average particle size of 2.5–3.5 mm, a water content of 90%–98%, a particle size recovery rate of 85%–95% after freeze-drying and rehydration, and a swelling rate of 700%–900%. The logarithmic decrease in viable bacterial count after four freeze-thaw cycles is no higher than 2.0, and the viable bacterial survival rate after 30 minutes of 254 nm ultraviolet irradiation is no less than 60%.

[0036] This invention also provides a method for preparing the microcapsules, comprising the following steps: (1) The composite microbial agent is mixed with sodium alginate solution to obtain sodium alginate-bacterial solution mixture; (2) The sodium alginate-bacterial solution mixture is dropped into calcium chloride solution for ionic cross-linking and solidification to obtain sodium alginate gel microspheres; (3) The sodium alginate gel microspheres are coated in chitosan solution to obtain sodium alginate-chitosan microcapsule composite bacterial agent.

[0037] In some specific embodiments, the final concentration of sodium alginate in the sodium alginate-bacterial solution mixture is 1.4%; The concentration of the calcium chloride solution is 1.5%; The concentration of the chitosan solution is 2.0%.

[0038] In some specific implementations, the ion crosslinking curing time is 20 minutes; The coating conditions are: 120 r / min oscillation coating for 20 to 40 min; When the sodium alginate-bacterial solution mixture is dripped into the calcium chloride solution for ionic cross-linking and solidification, the distance between the needle tip and the surface of the calcium chloride solution is controlled to be 5-10 cm.

[0039] The present invention also provides the application of the composite microbial agent or the microcapsule in promoting the low-temperature decomposition of rice straw.

[0040] In some specific implementations, the low temperature is 10°C.

[0041] The soil moisture content is 60%–80% of field capacity; when the microcapsules are used, the microcapsules are applied to the surface of rice straw or the interface between rice straw and soil; based on dry weight, each 5g of rice straw is treated with microcapsule compound bacterial agent equivalent to 0.5–2.0mL of compound bacterial solution for 30–60 days.

[0042] Example 1 Screening of candidate strains at low temperatures and determination of candidate components for complex bacterial systems Based on 49 candidate strains isolated in the laboratory previously, these strains were mainly isolated from natural environmental samples such as decomposed rice straw (collected from Yiyang, Hunan), camellia oleifera shells (collected from Changning, Hunan), decaying tree bark (collected from Jiulongjiang Forest Park, Hunan), and meadow soil (Nanshan National Park). Restrictive acclimatization screening was conducted at 5℃. After approximately two months of continuous cultivation, eight candidate strains capable of stable growth at 5℃ were obtained. Further analysis of the carboxymethyl cellulase, filter paper enzyme, and laccase activities of these candidate strains was performed, and inter-strain antagonism tests were used to screen for complex bacterial strain components.

[0043] Enzyme activity assays (Table 1) showed that the carboxymethyl cellulase (CMCase) activities of f206:SSTF1 and b362:SSTF3 were 42.04 U / mL and 41.81 U / mL, respectively, while BB-43 was 26.46 U / mL. Filter paper enzyme (FPA) activity was highest in BB-49 (22.43 U / mL), followed by b362:SSTF3 and BB-43. Lac activity was highest in f206:SSTF1 (64.92 U / mL). Antagonism assays showed that no significant growth inhibition bands were observed when b362:SSTF3, f206:SSTF1, BB-43, and BB-49 were cultured in pairs.

[0044] Table 1. Enzyme production characteristics of some candidate cryogenic degrading strains

[0045] Example 2 Optimization of compound bacterial strain ratio and verification of low-temperature degradation Using four bacterial strains—b362:SSTF3, f206:SSTF1, BB-43, and BB-49—as candidate components, a four-component mixing design was employed to optimize the ratio of the composite bacterial strain. The sum of the volume ratios of each component was set to 1, and the activities of carboxymethyl cellulase, filter paper enzyme, and laccase were used as response values. Candidate ratios were obtained through multi-response comprehensive optimization.

[0046] The candidate formulations were validated by degrading rice straw in conical flasks at 10℃ for 20 days. The results showed that ( Figure 3 The M4 group had the highest straw weight loss rate, at 17.18%. The volume ratio of the M4 group was 0.377:0.397:0.226:0, consisting of b362:SSTF3, f206:SSTF1, BB-43, and BB-49. Therefore, it was determined that a low-temperature composite bacterial system was formed by mixing b362:SSTF3, f206:SSTF1, and BB-43 in a bacterial liquid volume ratio of 0.377:0.397:0.226.

[0047] Table 2. Optimal composition and functional positioning of low-temperature composite bacterial strains

[0048] Sphingobacterium sp. b362:SSTF3 was deposited on June 4, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251237.

[0049] Paecilomycessp. f206:SSTF1 was deposited on March 20, 2025 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025548.

[0050] Flavobacterium sp. BB-43 was deposited on June 4, 2026 at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20261191.

[0051] Example 3 Preparation of compound bacterial solution b362:SSTF3 and BB-43 were inoculated separately into LB liquid medium and cultured with shaking at 10°C until the logarithmic growth phase. f206:SSTF1 was first activated on PDA medium and then inoculated into LB medium, and cultured with shaking at 10°C until the logarithmic growth phase. Before mixing, plate counts, spore counts, cell dry weight, or OD were used to measure the bacterial count. 600 Through conversion and other methods, the bacterial cultures were adjusted to the same order of magnitude, with an optimal number of culturable units of 1×10⁻⁶. 8 ~1×10 9 CFU / mL.

[0052] The b362:SSTF3 bacterial solution, f206:SSTF1 bacterial solution, and BB-43 bacterial solution were mixed at a volume ratio of 0.377:0.397:0.226 to obtain a composite bacterial solution. This composite bacterial solution can be used directly as a liquid bacterial agent or further used to prepare microcapsule composite bacterial agents.

[0053] Example 4 Preparation of sodium alginate-chitosan microcapsule composite bacterial agent Sterile sodium alginate solution (1.4 g / 100 mL), calcium chloride solution (1.5 g / 100 mL), and chitosan solution (2.0 g / 100 mL) were prepared separately, wherein the chitosan solution contained 1% acetic acid by volume. The b362:SSTF3 bacterial suspension, f206:SSTF1 bacterial suspension, and BB-43 bacterial suspension prepared in Example 3 were mixed at a volume ratio of 0.377:0.397:0.226 to obtain a composite bacterial suspension. The composite bacterial suspension was then mixed with the sodium alginate solution at a volume ratio of 1:10 to obtain a sodium alginate-composite bacterial suspension mixture, and the final concentration of sodium alginate in the mixture was brought to a preset concentration.

[0054] The sodium alginate-compound bacterial solution mixture is drawn into a disposable syringe, with the needle tip perpendicular to the surface of the calcium chloride solution. The distance between the needle tip and the liquid surface is controlled at 5-10 cm. The solution is then dripped dropwise into the calcium chloride solution at a constant rate of 0.5-1 s / drop. Upon entering the calcium chloride solution, the droplets undergo Ca2+ oxidation. 2+ Ionic crosslinking and solidification form sodium alginate gel microspheres.

[0055] After solidification, collect the gel microspheres and wash them 2-3 times with sterile distilled water to remove residual calcium chloride from the surface. Then, place the gel microspheres in a chitosan solution and shake at 100-150 rpm for 20-40 minutes to coat them, allowing chitosan to form a coating layer on the surface of the sodium alginate gel microspheres. After coating, collect the microspheres and wash them with sterile distilled water to obtain the sodium alginate-chitosan microcapsule composite bacterial agent.

[0056] Table 3 Preparation conditions of microencapsulated compound microbial agents

[0057] Example 5 Optimization of microcapsule preparation conditions Using microcapsule encapsulation efficiency and sphericity as the main evaluation indicators, the effects of sodium alginate concentration, calcium chloride concentration, curing time, and chitosan concentration on the microcapsule preparation effect were investigated. Single-factor experiments showed that sodium alginate concentration, calcium chloride concentration, curing time, and chitosan concentration all affected the microcapsule encapsulation efficiency, generally exhibiting a pattern of first increasing and then decreasing or stabilizing.

[0058] Among them, when the sodium alginate concentration increased from 0.5% to 1.1%, the encapsulation rate increased from 34.13% to 77.78%; after further increasing it to 2.0%, the encapsulation rate decreased to 32.53%. The calcium chloride concentration at 1.5% showed a relatively high encapsulation rate of 78.71%. When the curing time was extended from 5 min to 20 min, the encapsulation rate increased from 49.07% to 82.41%, but there was no significant increase after further extending it to 25–30 min.

[0059] Orthogonal experiments were conducted based on single-factor experiments. Range analysis showed that, under the experimental conditions, the influence of different factors on the encapsulation efficiency was in the order of sodium alginate concentration > calcium chloride concentration > chitosan concentration. Considering the encapsulation efficiency, mean analysis, and microcapsule spheroidization state, the optimal preparation conditions were determined to be 1.4% sodium alginate, 1.5% calcium chloride, 2.0% chitosan, and a curing time of 20 min.

[0060] Table 4. Summary of Optimization Results of Microcapsule Preparation Process

[0061] Example 6 Performance evaluation of microencapsulated compound microbial agents Microencapsulated compound bacterial agent was prepared under the preferred conditions determined in Example 5. The average wet particle size was determined by randomly selecting 30 microcapsules, measuring their diameters, and then averaging the results. Moisture content was calculated as "Moisture content = (Wet mass - Dry mass) / Wet mass × 100%". The particle size recovery rate after freeze-drying and rehydration was calculated as "Particle size recovery rate = Average particle size after rehydration / Average wet particle size before freeze-drying × 100%"; the swelling rate was calculated as "Swelling rate = (Rehydrated mass - Freeze-dried mass) / Freeze-dried mass × 100%".

[0062] In the freeze-thaw tolerance test, a freeze-thaw cycle consisted of freezing at -20℃ for 12 hours and thawing at 30℃ for 2 hours. This cycle was repeated four times before the viable cell count was determined, and the result was expressed as Δlog CFU = log10N0 - log10N. t This indicates the logarithmic decrease in viable bacterial count. In the UV tolerance test, the microcapsule compound bacterial agent and the free compound bacterial solution containing an equal amount of bacteria were irradiated under 254nm UV light for 30 minutes, and then the viable bacterial count was measured. The survival rate was expressed as N. t Calculate using / N0×100%.

[0063] The average particle size of the wet microcapsule bacterial agent is approximately 3.1 mm, with an initial moisture content of approximately 95%. After vacuum freeze-drying, the particles retain their intact morphology. After rehydration, the particle size recovery rate of the microcapsules reaches 90.29%, and the swelling rate is 838.20%, indicating that the microcapsules have good rehydration properties and water absorption and swelling capacity.

[0064] After four freeze-thaw cycles, the logarithmic decrease in viable cell count of the microcapsule composite bacterial agent was 1.67, while that of the free composite bacterial solution was 5.02, indicating that the microcapsule gel network can buffer the damage to the bacteria caused by repeated freeze-thaw cycles. After 30 minutes of UV irradiation, the viable cell count of the free composite bacterial solution increased from 5.27 × 10⁻⁶. 8 CFU / mL decreased to 3.60 × 10⁻⁶ 5 CFU / mL, survival rate approximately 0.07%; the viable count of the microcapsule compound bacterial agent increased from 5.33 × 10⁻⁶. 8 CFU / mL decreased to 3.53×10 8 The CFU / mL concentration and the survival rate of approximately 66% indicate that the microcapsule structure can reduce the damage to the bacteria caused by ultraviolet radiation.

[0065] Table 5 Key Performance Characteristics of Microencapsulated Compound Microbial Agents

[0066] Release tests showed that the microencapsulated compound bacterial agent exhibited a pattern of rapid release in the early stage and slower release in the later stage in sterile physiological saline: OD of the system in the initial 1-4 days of culture was [data missing]. 600 The rapid increase indicates that the live bacteria located on or near the surface of the microcapsule can be released earlier; the OD level is approximately 5–12 days later.600 The continued slow increase indicates that the bacteria inside the gel network can continue to diffuse outwards.

[0067] Scanning electron microscopy observation showed ( Figure 6 The microcapsules are nearly spherical in shape and retain their structure intact after freeze-drying, exhibiting distinct surface wrinkles. Fourier transform infrared spectroscopy indicates that sodium alginate, chitosan, and Ca... 2+ A complex network structure composed of hydrogen bonds, electrostatic interactions, and ionic cross-linking is formed between them, providing a carrier basis for the stable preservation and continuous release of bacterial cells after encapsulation.

[0068] Example 7 Application of microencapsulated compound microbial agents in promoting low-temperature decomposition of rice straw The soil samples were collected from the 0–20 cm topsoil layer of the rice-growing area, and plant debris and stones were removed before use. The rice straw samples were taken from mature rice plants, air-dried, leaves and panicles were removed, cut into approximately 5 cm pieces, and dried at 60℃ to constant weight.

[0069] A potted plant simulation system for returning rice to the field was established at 10℃ for 60 days. 200g of sieved paddy soil was placed in each sterilized polypropylene plastic box, and 5g of pretreated straw was evenly spread on the soil surface.

[0070] Set up four processing groups: Group S was a straw control group that was not inoculated with exogenous microbial agents; Group L consists of liquid bacterial agents inoculated with the same compound bacterial strain as in Example 3; Group M was inoculated with the microcapsule compound bacterial agent obtained in Example 5; Group CL was inoculated with a commercially available straw composting agent, whose main components are Bacillus spores, molds, actinomycetes, photosynthetic bacteria, lactic acid bacteria, and yeast. The product name is EM composting agent, purchased online.

[0071] Each group had four replicates. Group L received 1 mL of liquid inoculant diluted with sterile water and sprayed evenly onto the straw surface. Group M received a microcapsule compound inoculant equivalent to 1 mL of liquid inoculant. Group CL received the inoculant according to the instructions for use with a commercially available inoculant.

[0072] After cultivation (60 days later), the mechanical strength, chemical composition, scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, nutrient residue, and microbial community of the straw samples from each treatment were measured. The mechanical strength was measured using the three-point bending method, recording the flexural load of the straw at the moment of fracture.

[0073] The results showed that the flexural load of fresh straw was 286.33 g, which decreased to 145.50 g in group S, 110.68 g and 121.77 g in groups L and CL, respectively, and 53.21 g in group M, indicating that the microcapsule compound microbial agent can promote the disintegration of the straw skeleton.

[0074] The structural chemical composition analysis results showed that the contents of cellulose, hemicellulose, and lignin in the original straw were 38.08%, 22.76%, and 20.41%, respectively. After 60 days of low-temperature degradation, the contents of cellulose, hemicellulose, and lignin in group S were 32.41%, 17.04%, and 16.02%, respectively; in group L, they were 28.25%, 13.89%, and 11.78%, respectively; in group CL, they were 28.70%, 15.32%, and 15.12%, respectively; and in group M, they decreased to 19.00%, 13.62%, and 10.98%, respectively.

[0075] Table 6. Low-temperature decomposition effect of rice straw under different treatments

[0076] Scanning electron microscopy observation showed ( Figure 11 The surface structure of fresh straw is relatively intact. Group S only showed slight cracks and local peeling. The surface damage of Group L and Group CL was between that of Group S and Group M. Group M showed more complete surface damage, with large-area peeling, increased pores, and loose and collapsed fiber structure.

[0077] Fourier transform infrared spectroscopy analysis showed that ( Figure 12 The peak position changes in the OH stretching vibration region were more obvious in group M, indicating that the microcapsule compound microbial agent can weaken the hydrogen bonding in cellulose and hemicellulose; the carbonyl-related peak of hemicellulose ester was weakened or disappeared in the microbial agent treatment group, and the vibration peak of the lignin aromatic ring skeleton was weakened more significantly in group M.

[0078] X-ray diffraction analysis showed that ( Figure 13 The crystallinity index of fresh straw was 27.65%. After 60 days of low-temperature degradation, the crystallinity of groups S, CL, and L increased to 38.47%, 37.47%, and 39.60%, respectively, while that of group M was 34.71%, lower than that of groups S, CL, and L. This indicates that the microcapsule compound microbial agent not only promotes the removal of amorphous components but also promotes the destruction of crystalline cellulose regions.

[0079] The results of straw residual nutrient determination showed that ( Figure 14The total nitrogen, total phosphorus, and total potassium of fresh straw were 12.27 mg / g, 0.990 mg / g, and 18.28 mg / g, respectively. After 60 days of low-temperature degradation, the total nitrogen, total phosphorus, and total potassium of straw in group M decreased to 7.99 mg / g, 0.519 mg / g, and 4.11 mg / g, respectively, representing reductions of 34.95%, 47.62%, and 77.55% compared to fresh straw. This indicates that the microencapsulated compound microbial agent can promote the release of nutrients from straw.

[0080] Table 7. Changes in residual nutrients in straw under treatment with microencapsulated compound microbial agents (unit: mg / g)

[0081] Microbial community analysis results showed that treatment with the microencapsulated compound microbial agent maintained high bacterial and fungal diversity and regulated the microbial community structure on the straw surface. Within the fungal community, the relative abundance of some functional bacteria related to lignin degradation increased; the bacterial community composition also changed accordingly. These results indicate that under low-temperature return-to-field conditions, the microencapsulated compound microbial agent of this invention can promote rice straw decomposition through cell protection, sustained release, and community remodeling.

[0082] Compared with group S (without exogenous microbial agents), groups L, CL, and M all promoted the low-temperature decomposition of rice straw. Compared with group L (with the same strain of liquid microbial agents), group M exhibited higher activity retention under freeze-thaw cycles and UV stress, and further reduced straw flexural load and residual lignocellulose in a 10℃, 60-day simulated field return experiment, indicating a synergistic effect between the sodium alginate-chitosan microcapsule formulation and the specific low-temperature composite microbial strain. Compared with the commercially available straw decomposition agent group CL, group M showed stronger straw structure destruction ability and nutrient release promotion under low-temperature conditions.

[0083] The low-temperature composite microbial system and microcapsule composite microbial agent provided by this invention can be used for the low-temperature decomposition of rice straw in autumn and winter, and can also be used for the low-temperature biodegradation treatment of similar lignocellulose agricultural waste. The preparation method uses common materials such as sodium alginate, calcium chloride, and chitosan, and the process conditions are mild, which facilitates large-scale preparation and agricultural application.

[0084] The above embodiments are only used to illustrate the technical solution of the present invention. Those skilled in the art can make conventional adjustments to the culture conditions, wall material concentration, coating time, application dosage, and application environment without departing from the essence of the present invention, and all such adjustments should fall within the protection scope of the present invention.

[0085] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A compound microbial inoculant for promoting low-temperature decomposition of rice straw, characterized in that, Including Sphingosine Bacillus ( Sphingobacterium sp.) b362: SSTF3, Penicillium ( Paecilomyces sp.) f206: SSTF1 and Flavobacterium ( Flavobacterium sp.) BB-43; The *Sphingomonas* b362:SSTF3 was deposited on June 4, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251237. The *Paecilomyces f206:SSTF1* was deposited on March 20, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 2025548. The Flavobacterium BB-43 was deposited at the China Center for Type Culture Collection (CCTCC) on June 4, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20261191.

2. The compound microbial agent according to claim 1, characterized in that, The volume ratio of the bacterial culture of *Sphingosporobacter b362:SSTF3*, *Penicillium f206:SSTF1*, and *Flavobacterium BB-43* was 0.377:0.397:0.

226. The culture volume of *Sphingomonas b362:SSTF3*, *Penicillium f206:SSTF1*, and *Flavobacterium BB-43* is 1 × 10⁻⁶ units. 8 ~1×10 9 CFU / mL.

3. A microcapsule for promoting low-temperature decomposition of rice straw, characterized in that, Includes the composite microbial agent as described in claim 1 or 2.

4. The microcapsule according to claim 3, characterized in that, The wall material of the microcapsules includes sodium alginate, calcium chloride, and chitosan.

5. The method for preparing microcapsules as described in claim 3 or 4, characterized in that, Includes the following steps: (1) The composite microbial agent of claim 1 or 2 is mixed with sodium alginate solution to obtain sodium alginate-bacterial solution mixture; (2) The sodium alginate-bacterial solution mixture is dropped into calcium chloride solution for ionic cross-linking and solidification to obtain sodium alginate gel microspheres; (3) The sodium alginate gel microspheres are coated in chitosan solution to obtain sodium alginate-chitosan microcapsule composite bacterial agent.

6. The preparation method according to claim 5, characterized in that, The final concentration of sodium alginate in the sodium alginate-bacterial solution mixture is 1.4%. The concentration of the calcium chloride solution is 1.5%; The concentration of the chitosan solution is 2.0%.

7. The preparation method according to claim 5, characterized in that, The ion crosslinking curing time is 20 min; The coating conditions are: 120 r / min oscillation coating for 20 to 40 min; When the sodium alginate-bacterial solution mixture is dripped into the calcium chloride solution for ionic cross-linking and solidification, the distance between the needle tip and the surface of the calcium chloride solution is controlled to be 5-10 cm.

8. The application of the compound microbial agent as described in claim 1 or the microcapsule as described in claim 3 in promoting the low-temperature decomposition of rice straw.

9. The application according to claim 8, characterized in that, The low temperature is 10°C.

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