A highly stable, stress-resistant, synergistic microbial agent and its preparation method

Abstract

This invention discloses a highly stable, stress-resistant, and synergistically synergistic microbial inoculant and its preparation method. The method includes: constructing a composite microbial fermentation broth composed of primary degrading bacteria, auxiliary degrading bacteria, and stress-resistant colonizing bacteria through targeted enrichment and multi-dimensional functional screening; adding a protective agent to the concentrated fermentation broth and mixing it with a biochar / porous mineral framework carrier for adsorption; then mixing it with an encapsulation agent and a slow-release nutrient source, followed by extrusion, spheroidization, cross-linking solidification, and drying to produce granular inoculant. This invention utilizes a multiple coupling mechanism—synergistic construction of functional microbial communities, adsorption and protection by the framework carrier, enhanced stress resistance through encapsulation and cross-linking, and continuous supply of slow-release nutrients—to obtain an inoculant with advantages such as high viable cell count, good storage stability, strong tolerance to low water potential and wet-dry cycles, and high straw degradation rate. It is suitable for complex agricultural environments such as in-situ return of wheat straw to the field.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, and in particular to a highly stable, stress-resistant, synergistic microbial inoculant and its preparation method. Background Technology

[0002] In recent years, the in-situ return of wheat straw to the field has received widespread attention due to its ability to increase soil organic matter content, improve topsoil structure, and promote the resource utilization of agricultural waste. However, wheat straw contains a high proportion of cellulose, hemicellulose, and lignin, has a dense structure, and degrades slowly naturally. Under in-situ return conditions, it is prone to problems such as long decomposition cycles, competition for nitrogen with subsequent crops, insufficient local fermentation, and unstable soil ecological effects. To improve straw decomposition efficiency, existing technologies typically use single-agent, liquid, or ordinary solid microbial agents. Although these methods can promote straw degradation, they generally suffer from poor storage stability, insufficient adaptability to field environments, easy inactivation or loss of microorganisms after application, and short duration of action, making it difficult to meet the requirements of long-term effectiveness and stability for in-situ straw degradation.

[0003] Existing immobilized microbial agent technology has been applied in sludge treatment and organic waste degradation, typically using adsorption carriers, encapsulation materials, or slow-release nutrients to immobilize and protect the microorganisms. While this technology can increase the cell load and extend the action period, it still has shortcomings for in-situ degradation of wheat straw: on the one hand, existing agents mostly focus on a single degradation function, lacking the construction of composite microbial communities that take into account the degradation of the main component, survival under stress, and surface colonization; on the other hand, existing carriers and encapsulation systems are mostly designed for relatively stable environments, making it difficult to adapt to the complex conditions of low moisture, alternating wet and dry periods, and alkaline soils during straw return to the field, resulting in low activity retention rates and unstable synergistic degradation effects during storage and application. Therefore, developing a microbial agent with high stability, stress resistance, and synergistic effects has significant practical application value. Summary of the Invention

[0004] To address the aforementioned problems, this invention proposes a highly stable, stress-resistant, and synergistically effective microbial agent and its preparation method. By constructing a composite functional microbial community through targeted screening, and combining adsorption with a biochar / porous mineral framework carrier, stabilization with a protective agent, encapsulation and cross-linking, and slow-release nutrient supply, a granular microbial agent is prepared. This agent can improve storage stability and stress resistance survival ability, and enhance the continuous colonization and synergistic degradation of microorganisms during the in-situ return of wheat straw to the field.

[0005] This invention can be achieved through the following technical solutions: A method for preparing a highly stable, stress-resistant, synergistic microbial agent includes the following steps: S1. Collect soil samples of straw returned to the field, and conduct directional enrichment culture using wheat straw powder, sodium carboxymethyl cellulose and xylan as carbon sources to isolate candidate strains; screen the candidate strains for cellulose degradation ability, hemicellulose degradation ability, low water potential tolerance, dry-wet cycle tolerance, alkali tolerance and straw surface adhesion ability, and evaluate the compatibility between strains to construct a composite microbial fermentation broth composed of main degrading bacteria, auxiliary degrading bacteria and stress-resistant colonization bacteria; S2. Concentrate the composite microbial fermentation broth to 1 / 3-1 / 2 of its original volume, add a protective agent, and mix it evenly with the framework carrier to obtain an adsorption mixture; wherein the framework carrier is composed of biochar and porous mineral carrier. S3. Polyvinyl alcohol, sodium alginate, gelatin and starch are compounded in a mass ratio of 1:(3-5):(1-2):(1-2) and heated to dissolve to obtain an encapsulating agent; the adsorption mixture, encapsulating agent and slow-release nutrient source are mixed evenly and extruded into spherical particles with a particle size of 0.3-1.2 cm by a granulator to obtain initial particles, which are then placed in a saturated boric acid solution containing 2%-5% calcium chloride for cross-linking reaction to form gel particles, which are dried at 25-35℃ to obtain a highly stable stress-resistant synergistic microbial agent; wherein, the protective agent is trehalose and glycerol, and the slow-release nutrient source is a mixture of starch and polycaprolactone.

[0006] Preferably, the main degrading bacteria are cellulose-degrading bacteria, the auxiliary degrading bacteria are hemicellulose-degrading bacteria, and the stress-resistant colonization bacteria are strains that are resistant to low water potential, dry-wet cycles, alkali, and have the ability to adhere to the straw surface. The viable concentration of each strain in the compound microbial fermentation broth is 1×10⁹-5×10⁹ CFU / mL.

[0007] Preferably, the stress-resistant colonization bacteria are strains that simultaneously meet the following conditions: maintain growth capacity under PEG-simulated low water potential conditions, remain viable after at least three wet-dry alternation treatments, maintain growth capacity under pH 8.0-9.0 conditions, and are able to attach to the surface of sterilized wheat straw.

[0008] Preferably, the amount of trehalose added is 2%-6% of the mass of the compound microbial fermentation broth, and the amount of glycerol added is 0.5%-2% of the mass of the compound microbial fermentation broth.

[0009] Preferably, the mass ratio of biochar to porous mineral carrier is 1:(2-4), and the mass ratio of the skeleton carrier to the concentrated composite microbial fermentation broth is (5-8):1; the porous mineral carrier is one or more of vermiculite, zeolite, attapulgite, or diatomite.

[0010] Preferably, the mass ratio of starch to polycaprolactone is (30-50):(50-70).

[0011] Preferably, the mass ratio of the adsorption mixture, the encapsulating agent, and the slow-release nutrient source is 1:(1-3):(1-2), and the cross-linking reaction time is 24-48h.

[0012] Preferably, the moisture content of the microbial agent is 2%-8%.

[0013] Preferably, the microbial agent is added in granular form to the crushed wheat straw and the surrounding soil, with an addition amount of 0.5%-10% of the straw mass.

[0014] The beneficial effects of this invention are: This invention constructs a composite functional microbial community composed of primary degrading bacteria, auxiliary degrading bacteria, and stress-resistant colonizing bacteria through targeted screening. The bacteria are adsorbed and immobilized using a framework carrier composed of biochar and porous mineral carriers. Combined with the stabilizing effect of trehalose and glycerol as dual protective agents, a three-dimensional network gel structure is formed by cross-linking calcium chloride with a composite encapsulating agent of polyvinyl alcohol, sodium alginate, gelatin, and starch, thus embedding the bacteria inside the particles. At the same time, a mixture of starch and polycaprolactone is introduced as a slow-release nutrient source, achieving multi-level protection and functional synergy of the microbial agent. This microbial agent exhibits a high initial viable count and excellent stability during room temperature storage, significantly extending its shelf life. Its unique encapsulation and cross-linking system, working synergistically with the skeletal carrier, endows the agent with outstanding resistance to low water potential, wet-dry cycles, and alkali conditions, effectively ensuring the survival and colonization of microorganisms in the complex environment of straw return to the field. The continuous supply of slow-release nutrients maintains the long-term metabolic activity of the microbial community, promoting the synergistic degradation of cellulose and hemicellulose by the primary and secondary degrading bacteria, thereby significantly improving straw decomposition efficiency. The granular form facilitates application and is environmentally friendly. Attached Figure Description

[0015] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 The results included the viable cell retention rate of microbial agents, low water potential survival rate, wet-dry cycle survival rate, and straw weight loss rate. Detailed Implementation

[0016] The following provides a detailed description of the embodiments of the present invention: These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and processes. However, the scope of protection of the present invention is not limited to the following embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions.

[0017] Example 1: A method for preparing a highly stable, stress-resistant, synergistic microbial agent, comprising the following steps: S1. Collect soil samples with straw returned to the field. Use wheat straw powder, sodium carboxymethyl cellulose, and xylan as carbon sources for targeted enrichment culture to isolate candidate strains. Screen the candidate strains for cellulose degradation ability, hemicellulose degradation ability, low water potential tolerance, wet-dry cycle tolerance, alkali tolerance, and straw surface adhesion ability, and evaluate inter-strain compatibility. Alkali tolerance screening was performed at pH 8.0, and wet-dry cycle tolerance screening involved three cycles of alternating wet and dry treatments. Finally, primary degrading bacteria, auxiliary degrading bacteria, and stress-resistant colonization bacteria were screened, and a composite microbial fermentation broth was prepared, with a viable cell concentration of 1×10⁹ for each strain. CFU / mL; Take 40g of the compound microbial fermentation broth and concentrate it to 1 / 2 of its original volume to obtain 20g of concentrated compound microbial fermentation broth; Add 0.4g of trehalose and 0.1g of glycerol as protective agents and stir evenly; Weigh 100g of the skeleton carrier (composed of biochar and zeolite mixed in a mass ratio of 1:2) and mix it thoroughly with the concentrated compound microbial fermentation broth to obtain 120.5g of adsorption mixture; S2. Mix 20.08g polyvinyl alcohol, 60.25g sodium alginate, 20.08g gelatin and 20.08g starch, heat to 80℃ to dissolve, and obtain 120.5g encapsulation agent; S3. The adsorption mixture, encapsulating agent, and slow-release nutrient source (prepared by mixing starch and polycaprolactone in a mass ratio of 30:70) are mixed evenly in a mass ratio of 1:1:1. The mixture is then extruded and spherical granulated to form spherical particles with a particle size of 0.3 cm to obtain initial particles. The initial particles are then placed in a saturated boric acid solution containing 2% calcium chloride for cross-linking reaction for 24 h, and then dried at 25 °C. The moisture content of the obtained microbial agent is controlled to be 2% to obtain a highly stable, stress-resistant, synergistic microbial agent.

[0018] Example 2: A method for preparing a highly stable, stress-resistant, synergistic microbial agent, comprising the following steps: S1. Collect soil samples of straw returned to the field, and conduct targeted enrichment culture using wheat straw powder, sodium carboxymethyl cellulose, and xylan as carbon sources to isolate candidate strains. Screen the candidate strains for cellulose degradation ability, hemicellulose degradation ability, low water potential tolerance, wet-dry cycle tolerance, alkali tolerance, and straw surface adhesion ability, and evaluate inter-strain compatibility. Alkali tolerance screening was performed at pH 8.5, and wet-dry cycle tolerance screening involved three cycles of alternating wet and dry treatments. Finally, the main degrading bacteria, auxiliary degrading bacteria, and stress-resistant colonization bacteria were screened, and a compound microbial fermentation broth was prepared, with each strain having a viable cell concentration of 2.55 × 10¹⁰ CFU / mL. Take 48g of the compound microbial fermentation broth and concentrate it to 5 / 12 of its original volume to obtain 20g of concentrated compound microbial fermentation broth. Add 0.8g of trehalose and 0.25g of glycerol as protective agents and stir well. Weigh 130g of the skeleton carrier (composed of biochar and zeolite in a mass ratio of 1:3). The mixture was thoroughly mixed with the concentrated compound microbial fermentation broth to obtain 151.05g of adsorption mixture; S2. Polyvinyl alcohol, sodium alginate, gelatin and starch are mixed in a mass ratio of 1:4:1.5:1.5 and heated to 80℃ to dissolve, yielding 302.1g of encapsulating agent; S3. The adsorption mixture, encapsulating agent, and slow-release nutrient source (prepared by mixing starch and polycaprolactone at a mass ratio of 40:60) are mixed evenly at a mass ratio of 1:2:1.5. The mixture is then extruded and spherical granulated to form spherical particles with a particle size of 0.75 cm to obtain initial particles. The initial particles are then placed in a saturated boric acid solution containing 3.5% calcium chloride for cross-linking reaction for 36 h, and then dried at 30 °C. The moisture content of the obtained microbial agent is controlled to be 5% to obtain a highly stable, stress-resistant, synergistic microbial agent.

[0019] Example 3: A method for preparing a highly stable, stress-resistant, synergistic microbial agent, comprising the following steps: S1. Collect soil samples of straw returned to the field, and conduct directional enrichment culture using wheat straw powder, sodium carboxymethyl cellulose, and xylan as carbon sources to isolate candidate strains. Screen the candidate strains for cellulose degradation ability, hemicellulose degradation ability, low water potential tolerance, dry-wet cycle tolerance, alkali tolerance, and straw surface adhesion ability, and evaluate the compatibility between strains. Among them, the alkali tolerance screening condition is pH 9.0, and the dry-wet cycle tolerance screening is 3 dry-wet cycle treatments. Finally, the main degrading bacteria, auxiliary degrading bacteria, and stress-resistant colonization bacteria were screened, and a compound microbial fermentation broth was prepared, in which the viable cell concentration of each strain was 5×10¹⁰ CFU / mL. Take 60g of the compound microbial fermentation broth and concentrate it to 1 / 3 of the original volume to obtain 20g of concentrated compound microbial fermentation broth; add 1.2g of trehalose and 0.4g of glycerol as a protective agent and stir evenly; weigh 160g of the skeleton carrier (composed of biochar and zeolite in a mass ratio of 1:4) and mix it thoroughly with the concentrated compound microbial fermentation broth to obtain 181.6g of adsorption mixture; S2. Polyvinyl alcohol, sodium alginate, gelatin and starch were mixed in a mass ratio of 1:5:2:2 and heated to 80°C to dissolve, yielding 544.8g of encapsulating agent; S3. The adsorption mixture, encapsulating agent, and slow-release nutrient source (prepared by mixing starch and polycaprolactone in a mass ratio of 50:50) are mixed evenly in a mass ratio of 1:3:2. The mixture is then extruded and spherical granulated to form spherical particles with a particle size of 1.2 cm to obtain initial particles. Subsequently, the initial particles are placed in a saturated boric acid solution containing 5% calcium chloride for cross-linking reaction for 48 h, and then dried at 35 °C. The moisture content of the obtained microbial agent is controlled to be 8% to obtain a highly stable, stress-resistant, synergistic microbial agent.

[0020] Comparative Example 1: The difference between this comparative example and Example 1 is that no skeleton carrier is added.

[0021] A method for preparing a highly stable, stress-resistant, synergistic microbial agent includes the following steps: S1. Collect soil samples of straw returned to the field, and conduct directional enrichment culture using wheat straw powder, sodium carboxymethyl cellulose, and xylan as carbon sources to isolate candidate strains. Screen the candidate strains for cellulose degradation ability, hemicellulose degradation ability, low water potential tolerance, tolerance to wet-dry cycles, alkali tolerance, and straw surface adhesion ability, and evaluate inter-strain compatibility. Alkali tolerance screening was performed at pH 8.0, and wet-dry cycle tolerance screening involved three cycles of alternating wet and dry treatments. The main degrading bacteria, auxiliary degrading bacteria, and stress-resistant colonization bacteria were finally screened, and a compound microbial fermentation broth was prepared, with a viable cell concentration of 1×10⁹ CFU / mL for each strain. Take 40g of the compound microbial fermentation broth and concentrate it to half its original volume to obtain 20g of concentrated compound microbial fermentation broth. Add 0.4g of trehalose and 0.1g of glycerol as protective agents, stir well, and obtain the protected concentrated compound microbial fermentation broth. S2. Polyvinyl alcohol, sodium alginate, gelatin and starch are mixed in a mass ratio of 1:3:1:1 and heated to 80℃ to dissolve, yielding 20.5g of encapsulating agent; S3. The protected concentrated compound microbial fermentation broth, encapsulating agent, and slow-release nutrient source (prepared by mixing starch and polycaprolactone at a mass ratio of 30:70) are mixed evenly at a mass ratio of 1:1:1. The mixture is then extruded and spherical granulated to form spherical particles with a particle size of 0.3 cm to obtain initial particles. Subsequently, the initial particles are placed in a saturated boric acid solution containing 2% calcium chloride for cross-linking reaction for 24 h, and then dried at 25 °C. The moisture content of the obtained microbial agent is controlled to be 2% to obtain the microbial agent.

[0022] Comparative Example 2: The difference between this comparative example and Example 1 is that no slow-release nutrient source is added.

[0023] A method for preparing a highly stable, stress-resistant, synergistic microbial agent includes the following steps: S1. Collect soil samples with straw returned to the field. Use wheat straw powder, sodium carboxymethyl cellulose, and xylan as carbon sources for targeted enrichment culture to isolate candidate strains. Screen the candidate strains for cellulose degradation ability, hemicellulose degradation ability, low water potential tolerance, wet-dry cycle tolerance, alkali tolerance, and straw surface adhesion ability, and evaluate inter-strain compatibility. Alkali tolerance screening was performed at pH 8.0, and wet-dry cycle tolerance screening involved three cycles of alternating wet and dry treatments. Finally, primary degrading bacteria, auxiliary degrading bacteria, and stress-resistant colonization bacteria were screened, and a composite microbial fermentation broth was prepared, with a viable cell concentration of 1×10⁹ for each strain. CFU / mL; Take 40g of the compound microbial fermentation broth and concentrate it to 1 / 2 of its original volume to obtain 20g of concentrated compound microbial fermentation broth; Add 0.4g of trehalose and 0.1g of glycerol as protective agents and stir evenly; Weigh 100g of the skeleton carrier (composed of biochar and zeolite mixed in a mass ratio of 1:2) and mix it thoroughly with the concentrated compound microbial fermentation broth to obtain 120.5g of adsorption mixture; S2. Mix 20.08g polyvinyl alcohol, 60.25g sodium alginate, 20.08g gelatin and 20.08g starch, heat to 80℃ to dissolve, and obtain 120.5g encapsulation agent; S3. The adsorption mixture and the encapsulating agent are mixed evenly at a mass ratio of 1:1, and spherical particles with a particle size of 0.3 cm are formed by extrusion spheroidization granulation to obtain initial particles; then the initial particles are placed in a saturated boric acid solution containing 2% calcium chloride for cross-linking reaction for 24 h, and then dried at 25℃, controlling the water content of the obtained microbial agent to be 2% to obtain the microbial agent.

[0024] Comparative Example 3: The difference between this comparative example and Example 1 is that no inter-strain compatibility evaluation was performed.

[0025] A method for preparing a highly stable, stress-resistant, synergistic microbial agent includes the following steps: S1. Collect soil samples of straw returned to the field, and conduct directional enrichment culture using wheat straw powder, sodium carboxymethyl cellulose, and xylan as carbon sources to isolate candidate strains. Screen the candidate strains for cellulose degradation ability, hemicellulose degradation ability, low water potential tolerance, tolerance to wet-dry cycles, alkali tolerance, and straw surface adhesion ability. The alkali tolerance screening condition was pH 8.0, and the wet-dry cycle tolerance screening involved three cycles of alternating wet and dry treatments. Finally, the main degrading bacteria, auxiliary degrading bacteria, and stress-resistant colonization bacteria were screened, and a composite microbial fermentation broth was prepared, with a viable cell concentration of 1×10⁹ CFU / mL for each strain. Take 40g of the composite microbial fermentation broth and concentrate it to half its original volume to obtain 20g of concentrated composite microbial fermentation broth. Add 0.4g of trehalose and 0.1g of glycerol as protective agents and stir well. Weigh 100g of the framework carrier (composed of biochar and zeolite mixed in a mass ratio of 1:2) and mix it thoroughly with the concentrated composite microbial fermentation broth to obtain 120.5g of adsorption mixture. S2. Mix 20.08g polyvinyl alcohol, 60.25g sodium alginate, 20.08g gelatin and 20.08g starch, heat to 80℃ to dissolve, and obtain 120.5g encapsulation agent; S3. The adsorption mixture, encapsulating agent, and slow-release nutrient source (prepared by mixing starch and polycaprolactone in a mass ratio of 30:70) are mixed evenly in a mass ratio of 1:1:1. The mixture is then extruded and spherical granulated to form spherical particles with a particle size of 0.3 cm to obtain initial particles. The initial particles are then placed in a saturated boric acid solution containing 2% calcium chloride for cross-linking reaction for 24 h, and then dried at 25 °C. The moisture content of the obtained microbial agent is controlled to be 2% to obtain the microbial agent.

[0026] Performance testing 1 Initial viable count The initial viable count of microbial agents was tested according to the GB / T 20287-2006 standard.

[0027] 2. Viable bacteria retention rate during room temperature storage The microbial inoculant was dispensed into sealed aluminum foil bags and stored at 25°C in the dark. Samples were taken at 0, 30, 90, and 180 days, and the viable count was determined according to the "initial viable count" method. Viable count retention rate (%) = viable count after storage / initial viable count × 100%.

[0028] 3. Low water potential survival rate Microbial inoculant samples were taken, and after releasing the bacteria according to the viable cell count determination method, they were inoculated into a liquid culture system containing PEG-6000. A control group (0 MPa) and a stress group (-0.6 MPa) were set up. After culturing at 30℃ for 24 h, the viable cell counts were determined respectively. Low water potential survival rate = viable cell count in the stress group / initial viable cell count × 100%.

[0029] 4. Wet-dry cycle survival rate Take 100.0g of air-dried soil that has passed through a 2mm sieve, add sterile water to adjust to 60% water holding capacity, let stand for 12 hours, add microbial inoculant at a rate of 1.0g inoculant / 100g soil and mix thoroughly, immediately determine the initial viable count and record it as N0; incubate the sample at 30℃ for 24 hours and then air-dry it naturally until the soil moisture content drops below 20% water holding capacity, then add sterile water to restore it to 60% water holding capacity and let it stand for 12 hours, record this as one wet-dry cycle; after three consecutive wet-dry cycles, take a sample and determine the viable count according to the viable count determination method, record it as N1; the survival rate of the wet-dry cycle is calculated by the following formula: wet-dry cycle survival rate = N1 / N0 × 100%.

[0030] 5. Straw weight loss rate Wheat straw was cut into 2cm pieces and dried at 60℃ to constant weight. 5.0g was weighed as one sample and the initial dry weight was recorded. The straw and soil were placed together in a culture container, microbial inoculants were added, and the soil moisture content was adjusted to 60% water holding capacity. The mixture was cultured at 30℃ for 30 days. After the culture was completed, the straw was removed, surface soil particles were removed, and the straw was dried at 60℃ to constant weight. The dry weight after treatment was recorded. The straw weight loss rate was calculated as (initial dry weight - dry weight after treatment) / initial dry weight × 100%.

[0031] Table 1 Performance test results of microbial inoculants

[0032] As shown in Table 1, in terms of high stability, the viable cell retention rates of Examples 1-3 after 90 days of storage at 25℃ reached 64.3%, 78.9%, and 89.7%, respectively, which were significantly better than those of conventional unencapsulated or unprotected bacterial agents (usually less than 50%). This is attributed to the three-dimensional network gel structure formed by the encapsulating agent (polyvinyl alcohol / sodium alginate / gelatin / starch), the membrane protection effect of trehalose and glycerol, and the adsorption and fixation of biochar / porous mineral carrier. These three factors synergistically inhibited the metabolic decline of the bacteria during storage. In terms of stress resistance and synergistic effect: (1) Low water potential stress (-0.6MPa) Under PEG), the survival rate of Examples 1-3 was as high as 58.5%-85.6%, which is due to the low water potential resistance of the stress-resistant colonizing bacteria and the "micro water chamber" effect constructed by the encapsulation gel and the skeleton carrier, which effectively reduced the damage of osmotic stress to the bacteria; (2) After three cycles of dry and wet, the survival rate of Examples 1-3 was still 53.1%-81.3%, which proved that the encapsulation crosslinking system provided a physical barrier to protect the bacteria from mechanical damage and osmotic impact caused by drastic fluctuations in moisture; (3) The 30-day weight loss rate of straw was 27.6%-48.2%, which reflected the synergistic effect of the main degrading bacteria (cellulose degradation), auxiliary degrading bacteria (hemicellulose degradation) and stress-resistant colonizing bacteria. The stress-resistant colonizing bacteria ensured that the bacterial community survived in adversity and colonized on the surface of the straw, providing a "bridgehead" for the main / auxiliary degrading bacteria to continuously exert their degradation function, while the slow-release nutrient source (starch / polycaprolactone) continuously supplied carbon source to maintain the metabolic activity of the bacterial community. All three are indispensable.

[0033] In contrast, Comparative Example 1, without the addition of a scaffold carrier, showed a decrease in 90-day viable cell retention rate, low water potential survival rate, and wet-dry cycle survival rate to 41.8%, 34.7%, and 29.6%, respectively, representing decreases of 22.5, 23.8, and 23.5 percentage points compared to Example 1. This indicates that the absence of the scaffold carrier (biochar + porous minerals) resulted in a lack of adsorption support and microenvironment isolation for the bacteria, making them highly susceptible to inactivation under storage and abiotic stress, severely impairing their stability and resilience. Comparative Example 2, without the addition of a slow-release nutrient source, showed a decrease in 90-day viable cell retention rate, low water potential survival rate, and wet-dry cycle survival rate to 55.2%, 46.8%, and 41.4%, respectively. In particular, the straw weight loss rate decreased to 16.3%, only 59% of that in Example 1. This demonstrates that the lack of a slow-release nutrient source leads to metabolic decline in the bacterial community due to carbon source depletion during straw degradation, preventing the sustained synergistic effect—even if the bacteria survive, the lack of nutrient supply cannot maintain efficient degradation activity. Comparative Example 3 did not undergo interstrain compatibility evaluation. The 90-day viable cell retention rate, low water potential survival rate, and wet-dry cycle survival rate were 57.1%, 44.1%, and 39.8%, respectively, and the straw weight loss rate was 22.7%, all of which were significantly lower than those in Example 1. This demonstrates that there may be antagonistic effects between strains that have not undergone compatibility screening, which weakens the synergistic survival and degradation ability of the bacterial community under stress. Even if each individual strain has stress resistance, it is impossible to form an effective functional coupling.

[0034] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a high-stability stress-resistant synergistic microbial inoculant, characterized in that, Includes the following steps: S1. Collect soil samples of straw returned to the field, and conduct directional enrichment culture using wheat straw powder, sodium carboxymethyl cellulose and xylan as carbon sources to isolate candidate strains; Candidate strains were screened for their cellulose degradation ability, hemicellulose degradation ability, low water potential tolerance, dry-wet cycle tolerance, alkali tolerance, and straw surface adhesion ability. The compatibility between strains was evaluated, and a composite microbial fermentation broth consisting of main degrading bacteria, auxiliary degrading bacteria, and stress-resistant colonization bacteria was constructed. S2. Concentrate the composite microbial fermentation broth to 1 / 3-1 / 2 of its original volume, add a protective agent, and mix it evenly with the framework carrier to obtain an adsorption mixture; wherein the framework carrier is composed of biochar and porous mineral carrier. S3. Polyvinyl alcohol, sodium alginate, gelatin and starch are compounded in a mass ratio of 1:(3-5):(1-2):(1-2) and heated to dissolve to obtain an encapsulating agent; the adsorption mixture, encapsulating agent and slow-release nutrient source are mixed evenly and extruded into spherical particles with a particle size of 0.3-1.2 cm by a granulator to obtain initial particles, which are then placed in a saturated boric acid solution containing 2%-5% calcium chloride for cross-linking reaction to form gel particles, which are dried at 25-35℃ to obtain a highly stable stress-resistant synergistic microbial agent; wherein, the protective agent is trehalose and glycerol, and the slow-release nutrient source is a mixture of starch and polycaprolactone.

2. The method for preparing the high-stability stress-resistant synergistic microbial inoculant according to claim 1, characterized in that, The main degrading bacteria are cellulose-degrading bacteria, the auxiliary degrading bacteria are hemicellulose-degrading bacteria, and the stress-resistant colonization bacteria are strains that are resistant to low water potential, dry-wet cycles, alkali, and have the ability to adhere to the straw surface. The viable cell concentration of each strain in the compound microbial fermentation broth is 1×10⁹-5×10⁹ CFU / mL.

3. The preparation method of the highly stable, stress-resistant, synergistic microbial agent according to claim 1, characterized in that, The stress-resistant colonization bacteria are strains that simultaneously meet the following conditions: maintain growth ability under PEG-simulated low water potential conditions, remain viable after at least 3 dry-wet alternation treatments, maintain growth ability under pH 8.0-9.0 conditions, and are able to attach to the surface of sterilized wheat straw.

4. The preparation method of the highly stable, stress-resistant, synergistic microbial agent according to claim 1, characterized in that, The amount of trehalose added is 2%-6% of the mass of the compound microbial fermentation broth, and the amount of glycerol added is 0.5%-2% of the mass of the compound microbial fermentation broth.

5. The method for preparing the highly stable, stress-resistant, synergistically enhancing microbial agent according to claim 1, characterized in that, The mass ratio of biochar to porous mineral carrier is 1:(2-4), and the mass ratio of the skeleton carrier to the concentrated composite microbial fermentation broth is (5-8):1; the porous mineral carrier is one or more of vermiculite, zeolite, attapulgite, or diatomite.

6. The method for preparing the highly stable, stress-resistant, synergistically synergistic microbial agent according to claim 1, characterized in that, The mass ratio of starch to polycaprolactone is (30-50):(50-70).

7. The method for preparing the highly stable, stress-resistant, synergistically enhancing microbial agent according to claim 1, characterized in that, The mass ratio of the adsorption mixture, the encapsulating agent, and the slow-release nutrient source is 1:(1-3):(1-2), and the cross-linking reaction time is 24-48h.

8. The method for preparing the highly stable, stress-resistant, synergistically synergistic microbial agent according to claim 1, characterized in that, The moisture content of the microbial agent is 2%-8%.

9. The method for preparing the highly stable, stress-resistant, synergistically potent microbial agent according to claim 1, characterized in that, The microbial agent is added in granular form to the crushed wheat straw and the surrounding soil, at a rate of 0.5%-10% of the straw mass.

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