Saline-alkaline tolerant compound microbial agent and application thereof

Abstract

The invention discloses a saline-alkaline tolerant compound microbial agent and application thereof, and particularly relates to the technical field of microbial soil treatment. The microbial agent comprises Klebsiella, Enterobacter hormaechei and humic acid, the ratio of the components is as follows: Klebsiella: Enterobacter hormaechei: humic acid = 1: (0.5-2): (1-3), and both Klebsiella and Enterobacter hormaechei are obtained by fermentation culture. According to the present invention, the specific pH gradient is adopted for the Klebsiella and the Enterobacter hormaechei, and the segmented fermentation process combining the specific pH gradient with the corresponding gradually-increased sodium chloride concentration is adopted, such that the strain is effectively subjected to the directional domestication so as to maintain the activity and the function in the high-salt and high-alkali extreme environment.

Description

Technical Field

[0001] This invention relates to the field of microbial soil improvement technology, specifically to a salt- and alkali-tolerant compound microbial agent and its application. Background Technology

[0002] Soil compaction refers to the phenomenon where the surface layer of soil becomes compacted due to various reasons, resulting in a decrease in soil organic matter content and poor structure. Under the influence of external factors such as irrigation or rainfall, the soil structure is damaged and aggregates are dispersed. After drying, the soil surface hardens due to cohesive forces, reducing soil porosity, aeration and water permeability, and tillage performance. It also reduces the content of available nutrients, thereby affecting the normal growth and development of crops and leading to a decrease in yield.

[0003] With economic development and the widespread application of agricultural mechanization, the compaction of soil by agricultural machinery has subjected it to excessive external loads, damaging its original loose soil aggregate structure. Long-term shallow tillage has led to increasing compaction of the soil below the topsoil layer, disrupting the biological system and soil structure. Excessive tillage damages soil capillaries, enhancing capillary action and drastically reducing the soil's water retention and aeration capacity. Furthermore, the pursuit of higher yields through the application of large amounts of chemical fertilizers has disrupted the soil's nutrient structure, leading to a more homogenous soil structure and accelerating soil degradation. For example, excessive nitrogen fertilizer application causes rapid decomposition of soil organic matter, and excessive phosphate fertilizer application results in the formation of insoluble phosphate precipitates from excess phosphate and calcium and magnesium cations in the soil, further damaging the soil's composition and nutrient structure. All of these factors exacerbate soil compaction.

[0004] For soil compaction, various methods are commonly used, including mechanical treatment, inorganic and organic fertilizer application, and microbial inoculant treatment. Microbial inoculants, due to their harmlessness and high efficiency, are currently the best treatment for soil compaction. However, most current microbial inoculants are single-ingredient agents or simple compound agents. For example, Chinese patent CN116535269A uses heat-tolerant phosphate-solubilizing bacteria and Bacillus subtilis, while Chinese patent CN114686238A uses Bacillus amyloliquefaciens liquid and Bacillus subtilis liquid. However, these patented microbial inoculants are not tolerant to saline-alkali conditions, limiting their further application. Chinese patents CN113186117A and CN114410509A both mention that Klebsiella pneumoniae has a certain degree of saline-alkali tolerance, but conventionally cultured microorganisms do not achieve the expected saline-alkali tolerance in practical applications, and single-ingredient agents are not effective in controlling soil compaction, further limiting their application.

[0005] Therefore, finding a suitable compound microbial agent with high salt and alkali tolerance for soil compaction control is an urgent problem to be solved. Summary of the Invention

[0006] Therefore, the present invention provides a salt- and alkali-resistant composite microbial agent and its application to solve the problems in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: According to one aspect of the present invention, a salt- and alkali-resistant compound microbial agent is provided, the microbial agent comprising Klebsiella pneumoniae, Enterobacter holmium hominis and humic acid, wherein the ratio of each component is Klebsiella pneumoniae: Enterobacter holmium hominis: humic acid = 1:0.5~2:1~3, wherein both Klebsiella pneumoniae and Enterobacter holmium hominis are obtained by fermentation culture.

[0008] As an example, the preferred ratio of Klebsiella pneumoniae: Enterobacter holmium: humic acid is 1:1:2.

[0009] During the granulation process of the salt- and alkali-resistant composite microbial agent, auxiliary materials are added in the following ratio: Klebsiella pneumoniae: Enterobacter hominis: humic acid: auxiliary materials = 1:0.5~2:1~3:1~3. The auxiliary materials are wood ash and vermiculite powder, with the ratio of wood ash to vermiculite powder being 1~2:1.

[0010] Furthermore, the fermentation culture method for Klebsiella includes: firstly, controlling the reaction conditions to perform a preliminary culture of Klebsiella to obtain a primary seed culture; then, performing a secondary culture of the primary seed culture to obtain a secondary seed culture; finally, performing a staged fermentation of the secondary seed culture under different pH gradients to obtain Klebsiella fermentation broth; wherein the pH gradient is 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, 9.5~10.0, and the culture time for each stage is 5~10 hours.

[0011] Furthermore, the preliminary culture conditions are as follows: shake culture in an incubator at a temperature of 25~40℃ for 18~30h, with the rotation speed controlled at 150~200rpm.

[0012] Furthermore, the secondary culture conditions are: temperature 25~40℃, rotation speed 150~200rpm, pH 6.5~7.5, dissolved oxygen 20-50%, and culture time 18~30h.

[0013] Furthermore, the sodium chloride concentrations corresponding to the pH gradients are 8~15 g / L, 15~30 g / L, 30~50 g / L, 50~80 g / L, and 80~100 g / L.

[0014] Furthermore, the fermentation culture method for *Enterobacter holmieae* includes: firstly, controlling the reaction conditions to perform a preliminary culture of *Enterobacter holmieae* to obtain a primary seed culture; then, performing a secondary culture on the primary seed culture to obtain a secondary seed culture; finally, performing a staged fermentation of the secondary seed culture under different pH gradients to obtain *Enterobacter holmieae* fermentation broth; wherein the pH gradient is 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, 9.5~10.0, and the culture time for each stage is 5~10 hours.

[0015] Furthermore, the preliminary culture conditions are as follows: shake culture in an incubator at a temperature of 25~40℃ for 18~30h, with the rotation speed controlled at 150~200rpm.

[0016] Furthermore, the secondary culture conditions are: temperature 25~40℃, rotation speed 150~200rpm, dissolved oxygen 20-50%, and culture time 18~30h.

[0017] Furthermore, the sodium chloride concentrations corresponding to the pH gradients are 8~15 g / L, 15~30 g / L, 30~50 g / L, 50~80 g / L, and 80~100 g / L.

[0018] Secondary seed culture was inoculated into the fermenter at an inoculation rate of 10-20% of the total fraction.

[0019] According to another aspect of the present invention, a method for preparing the salt- and alkali-resistant composite microbial agent as described above includes: Step 1: Centrifuge the Klebsiella fermentation broth and the Enterobacter hopnea fermentation broth separately, adjust the solid content of the bacterial broth to 50-80%, and add 0.5-15% of the desiccant added to the bacterial broth to obtain Klebsiella bacterial slurry and Enterobacter hopnea bacterial slurry, respectively. Step 2: Mix the Klebsiella pneumoniae slurry, Enterobacter holmella slurry, humic acid and excipients from Step 1 in a certain proportion and granulate to obtain solid particles; Step 3: Vacuum dry the solid particles from Step 2 to obtain the composite microbial agent.

[0020] As an example, the preferred desiccant is one or more of galactose, trehalose, dimethyl sulfoxide, and gelatin.

[0021] As an example, the preferred drying temperature is 30~45℃ and the drying time is 5~15h.

[0022] The present invention has the following advantages: This invention employs a segmented fermentation process with a specific pH gradient and correspondingly increasing sodium chloride concentration to effectively acclimate Klebsiella and Enterobacter hominis, enabling them to maintain activity and function under extreme high-salt and high-alkali environments. This far exceeds the tolerance limits of conventionally cultured microorganisms.

[0023] This invention involves the targeted domestication of Klebsiella pneumoniae and Enterobacter hominis, followed by a combination with humic acid. This combination enhances the strains' tolerance to salinity and alkali, with the two strains working synergistically to improve soil structure. Furthermore, the humic acid, acting not only as a carrier and binder, but also by improving soil structure, enhancing water and fertilizer retention, stimulating microbial activity, and synergistically promoting the formation and stability of soil aggregates. The combination of these three agents more effectively decomposes organic matter in the soil, promotes mineral dissolution, and secretes extracellular polysaccharides to bind soil particles, thereby significantly improving soil porosity, aeration, water permeability, tillage performance, and nutrient availability. This fundamentally alleviates soil compaction problems, demonstrating superior effects compared to single-agent or simple compound microbial agents.

[0024] The compound microbial agent of the present invention helps to break up the compacted layer caused by long-term improper farmland management by improving soil structure and microbial activity, thus creating a more favorable environment for root growth.

[0025] The outstanding salt and alkali resistance of the compound microbial agent of this invention makes it particularly suitable for soils that are salinized, have secondary salinization, or have become compacted due to long-term application of chemical fertilizers, thus greatly expanding the application range of microbial agents.

[0026] The compound microbial agent of this invention is harmless, highly efficient, and environmentally friendly, meeting the requirements of green agriculture and sustainable soil management. By improving compacted soil through biological means, it reduces excessive reliance on mechanical deep tillage and chemical amendments.

[0027] The compound microbial agent of the present invention is highly effective in treating soil compaction caused by factors such as calcium sulfate accumulation and organic matter decline. It is especially suitable for saline-alkali compacted soils that are difficult to treat effectively with existing technologies, and solves the long-standing technical bottleneck in this field. It has broad application prospects and important practical value. Detailed Implementation

[0028] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Klebsiella pneumoniae: China General Microbiological Culture Collection Center CGMCC1.839; Enterobacter hopterii: China General Microbiological Culture Collection Center CGMCC1.10608; Humic acid: Guangdong Best Chemical Technology Co., Ltd., CAS: 1415-93-6; Wood ash: Shuyang Linzhiya Horticulture Co., Ltd., batch number: 03215; Vermiculite powder: Hebei Hongyao Mineral Products Processing Co., Ltd., batch number: 200; Galactose: Shanghai Maclean Biochemical Technology Co., Ltd., CAS: 59-23-4; Dimethyl sulfoxide: Shanghai Maclean Biochemical Technology Co., Ltd., CAS: 67-68-5; Gelatin: Comio Chemical Reagent Co., Ltd., CAS: 9000-70-8; Trehalose: Comio Chemical Reagent Co., Ltd., CAS: 6138-23-4.

[0030] Example 1 This embodiment provides a salt- and alkali-resistant compound microbial agent: 1. The fermentation method of Klebsiella pneumoniae is as follows: 1) Klebsiella pneumoniae was inoculated into seed culture medium and cultured in an incubator at 30°C with shaking for 24 hours to obtain primary seed liquid, with the rotation speed controlled at 180 rpm; 2) The seed solution obtained in step 1) is inoculated into the primary seed tank at an inoculation rate of 10% by volume. The culture conditions of the primary seed tank are: temperature 30℃, rotation speed 180rpm, pH 7.0, dissolved oxygen 40%, and cultured for 24h to obtain the secondary seed solution. 3) The secondary seed culture obtained in step 2) was inoculated into a fermenter at an inoculum of 15%. The fermenter was cultured at 30°C and 180 rpm, and the fermentation was carried out in stages at different pH gradients to obtain the fermentation broth. The pH gradients were 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, and 9.5~10.0, with each stage culture time being 5h, 6h, 6h, 8h, and 10h, respectively. Corresponding to different pH gradients, sodium chloride concentrations of 10 g / L, 20 g / L, 40 g / L, 60 g / L, and 80 g / L were used to obtain Klebsiella pneumoniae liquid inoculum.

[0031] 2. The fermentation method for Enterobacter hooligans is as follows: 1) Inoculate Enterobacter holmie into seed culture medium and culture in an incubator at 28℃ with shaking for 28h to obtain primary seed culture, and control the rotation speed at 160rpm; 2) The seed solution obtained in step 1) is inoculated into the primary seed tank at an inoculation rate of 15% by volume. The culture conditions of the primary seed tank are: temperature 28℃, rotation speed 160rpm, pH 7.2, dissolved oxygen 35%, and culture for 28h to obtain the secondary seed solution. 3) The secondary seed culture obtained in step 2) was inoculated into a fermenter at an inoculum of 15%. The fermentation conditions were 28°C and 160 rpm. Fermentation was carried out in stages at different pH gradients to obtain fermentation broth. The pH gradients were 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, and 9.5~10.0. The culture times for each stage were 5h, 6h, 6h, 8h, and 10h, respectively. The sodium chloride concentrations corresponding to the different pH gradients were 10g / L, 20g / L, 40g / L, 60g / L, and 80g / L, respectively, to obtain the liquid inoculum of Enterobacter holmieae.

[0032] 3. The preparation method of the compound microorganism is as follows: 1) Centrifuge the Klebsiella fermentation broth and Enterobacter hominis separately, adjust the solid content of the bacterial broth to 60%, add 10% desiccant to the adjusted bacterial broth to obtain bacterial slurry. The desiccant is galactose: dimethyl sulfoxide: gelatin = 1:1:1. 2) Mix the Klebsiella pneumoniae slurry, Enterobacter holmella slurry, humic acid and excipients obtained in step 1) in a mass ratio of 1:1:2:2 and granulate to obtain solid particles. The excipients are wood ash and vermiculite powder, with the ratio of wood ash to vermiculite powder being 2:1. 3) The solid particles obtained in step 2) are vacuum dried at a temperature of 30°C for 10 hours to obtain the composite microorganisms.

[0033] Example 2 This embodiment provides a salt- and alkali-resistant compound microbial agent: 1. The fermentation method of Klebsiella pneumoniae is as follows: 1) Klebsiella pneumoniae was inoculated into seed culture medium and cultured in an incubator at 30°C with shaking for 24 hours to obtain primary seed liquid, with the rotation speed controlled at 180 rpm; 2) The seed solution obtained in step 1) is inoculated into the primary seed tank at an inoculation rate of 10% by volume. The culture conditions of the primary seed tank are: temperature 25℃, rotation speed 200rpm, pH 7.5, dissolved oxygen 50%, and culture for 30h to obtain the secondary seed solution. 3) The secondary seed culture obtained in step 2) was inoculated into a fermenter at an inoculum of 10%. The fermentation conditions were 25°C and 200 rpm. Fermentation was carried out in stages at different pH gradients to obtain fermentation broth. The pH gradients were 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, and 9.5~10.0. The culture times for each stage were 5h, 6h, 6h, 8h, and 10h, respectively. For different pH gradients, sodium chloride concentrations of 8 g / L, 15 g / L, 30 g / L, 50 g / L, and 80 g / L were used to obtain Klebsiella liquid inoculum.

[0034] 2. The fermentation method for Enterobacter hooligans is as follows: 1) Inoculate Enterobacter holmie into seed culture medium and culture in an incubator at 25°C with shaking for 30 hours to obtain primary seed culture, and control the rotation speed at 200 rpm; 2) The seed solution obtained in step 1) is inoculated into the primary seed tank at an inoculation rate of 10% by volume. The culture conditions of the primary seed tank are: temperature 25℃, rotation speed 200rpm, pH 7.5, dissolved oxygen 50%, and culture for 30h to obtain the secondary seed solution. 3) The secondary seed culture obtained in step 2) was inoculated into a fermenter at an inoculum of 10%. The fermentation conditions were 25°C and 200 rpm. Fermentation was carried out in stages at different pH gradients to obtain fermentation broth. The pH gradients were 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, and 9.5~10.0. The culture times for each stage were 5h, 6h, 6h, 8h, and 10h, respectively. The sodium chloride concentrations corresponding to the different pH gradients were 8g / L, 15g / L, 30g / L, 50g / L, and 80g / L, respectively, to obtain the liquid inoculum of Enterobacter holmieae.

[0035] 3. The preparation method of the compound microorganism is as follows: 1) Centrifuge the Klebsiella fermentation broth and Enterobacter hominis separately, adjust the solid content of the bacterial broth to 60%, add 10% desiccant to the adjusted bacterial broth to obtain bacterial slurry. The desiccant is galactose: dimethyl sulfoxide: gelatin = 1:1:1. 2) The Klebsiella pneumoniae slurry, Enterobacter holmella slurry, humic acid, and excipients obtained in step 1) are mixed and granulated in a mass ratio of 1:0.8:1.5:1.5 to obtain solid particles. The excipients are wood ash and vermiculite powder, with a wood ash:vermiculite powder ratio of 1:1. 3) The solid particles obtained in step 2) are vacuum dried at a temperature of 45°C for 5 hours to obtain the composite microorganisms.

[0036] Example 3 This embodiment provides a salt- and alkali-resistant compound microbial agent: 1. The fermentation method of Klebsiella pneumoniae is as follows: 1) Klebsiella pneumoniae was inoculated into seed culture medium and cultured in an incubator at 30°C with shaking for 24 hours to obtain primary seed liquid, with the rotation speed controlled at 180 rpm; 2) The seed solution obtained in step 1) is inoculated into the primary seed tank at an inoculation amount of 20% by volume. The culture conditions of the primary seed tank are: temperature 40℃, rotation speed 150rpm, pH 6.5, dissolved oxygen 20%, and cultured for 18h to obtain the secondary seed solution. 3) The secondary seed culture obtained in step 2) was inoculated into a fermenter at an inoculum of 20% of the total volume. The fermentation conditions were 40℃ and 150 rpm. Fermentation was carried out in stages at different pH gradients to obtain the fermentation broth. The pH gradients were 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, and 9.5~10.0. The culture times for each stage were 5h, 6h, 6h, 8h, and 10h, respectively. For different pH gradients, sodium chloride concentrations of 15 g / L, 30 g / L, 50 g / L, 80 g / L, and 100 g / L were used to obtain Klebsiella liquid inoculum.

[0037] 2. The fermentation method for Enterobacter hooligans is as follows: 1) Inoculate Enterobacter holmie into seed culture medium and culture in an incubator at 40℃ with shaking for 18h to obtain primary seed culture, and control the rotation speed at 150rpm; 2) The seed solution obtained in step 1) is inoculated into the primary seed tank at an inoculation amount of 20% by volume. The culture conditions of the primary seed tank are: temperature 40℃, rotation speed 150rpm, pH 6.5, dissolved oxygen 20%, and cultured for 18h to obtain the secondary seed solution. 3) The secondary seed culture obtained in step 2) was inoculated into a fermenter at an inoculum of 20% of the total volume. The fermentation conditions were 40℃ and 150 rpm. Fermentation was carried out in stages at different pH gradients to obtain fermentation broth. The pH gradients were 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, and 9.5~10.0, with each stage having a culture time of 5h, 6h, 6h, 8h, and 10h, respectively. The sodium chloride concentrations corresponding to the different pH gradients were 15g / L, 30g / L, 50g / L, 80g / L, and 100g / L, respectively, to obtain a liquid inoculum of Enterobacter holmieae.

[0038] 3. The preparation method of the compound microorganism is as follows: 1) Centrifuge the Klebsiella fermentation broth and Enterobacter hominis separately, adjust the solid content of the bacterial broth to 60%, add 10% desiccant to the adjusted bacterial broth to obtain bacterial slurry. The desiccant is galactose: dimethyl sulfoxide: gelatin = 1:1:1. 2) Mix the Klebsiella pneumoniae slurry, Enterobacter holmella slurry, humic acid, and excipients obtained in step 1) in a mass ratio of 1:1.5:3:3 and granulate to obtain solid particles. The excipients are wood ash and vermiculite powder, with a wood ash:vermiculite powder ratio of 1.5:1. 3) The solid particles obtained in step 2) are vacuum dried at a temperature of 30°C for 15 hours to obtain the composite microorganisms.

[0039] Comparative Example 1 This comparative example provides a salt- and alkali-resistant compound microbial agent: 1. The fermentation method of Klebsiella pneumoniae is as follows: 1) Klebsiella pneumoniae was inoculated into seed culture medium and cultured in an incubator at 30°C with shaking for 24 hours to obtain primary seed liquid, with the rotation speed controlled at 180 rpm; 2) The seed solution obtained in step 1) is inoculated into the primary seed tank at an inoculation rate of 10% by volume. The culture conditions of the primary seed tank are: temperature 30℃, rotation speed 180rpm, pH 7.0, dissolved oxygen 40%, and cultured for 24h to obtain the secondary seed solution.

[0040] 3) The secondary seed culture obtained in step 2) was inoculated into a fermenter at an inoculation amount of 15%. The fermenter was cultured at a temperature of 30℃, a rotation speed of 180 rpm, a pH of 7.2, and a sodium chloride concentration of 10 g / L for 35 h to obtain Klebsiella liquid inoculum.

[0041] 2. The fermentation method for Enterobacter hooligans is as follows: 1) Inoculate Enterobacter holmie into seed culture medium and culture in an incubator at 28℃ with shaking for 28h to obtain primary seed culture, and control the rotation speed at 160rpm; 2) The seed solution obtained in step 1) is inoculated into the primary seed tank at an inoculation rate of 15% by volume. The culture conditions of the primary seed tank are: temperature 28℃, rotation speed 160rpm, pH 7.2, dissolved oxygen 35%, and culture for 28h to obtain the secondary seed solution.

[0042] 3) The secondary seed culture obtained in step 2) was inoculated into a fermenter at an inoculation amount of 15%. The fermenter was cultured for 35 hours at a temperature of 28°C, a rotation speed of 160 rpm, a pH of 7.2, and a sodium chloride concentration of 10 g / L to obtain Klebsiella liquid inoculum.

[0043] Everything else is the same as in Example 1.

[0044] Comparative Example 2 The microbial agents used in this comparative example contain only Klebsiella pneumoniae and do not contain Enterobacter holmium or humic acid. Unlike Example 1, this comparative example does not include fermentation with Enterobacter holmieae.

[0045] The preparation method of the composite microorganisms in this comparative example is as follows: 1) Centrifuge the Klebsiella fermentation broth, adjust the solid content of the broth to 60%, add 10% desiccant to the adjusted broth to obtain the bacterial slurry. The desiccant is galactose: dimethyl sulfoxide: gelatin = 1:1:1. 2) Mix the Klebsiella pneumoniae slurry and excipients obtained in step 1) at a mass ratio of 4:2 and granulate to obtain solid particles. The excipients are wood ash and vermiculite powder, with a wood ash:vermiculite powder ratio of 2:1. 3) The solid particles obtained in step 2) are vacuum dried at a temperature of 30°C for 10 hours to obtain the composite microorganisms.

[0046] Comparative Example 3 The microbial agent used in this comparative example contains only *Enterobacter johnsonii* and does not contain *Klebsiella pneumoniae* or humic acid. Unlike Example 1, this comparative example does not include Klebsiella fermentation.

[0047] The preparation method of the composite microorganisms in this comparative example is as follows: 1) Centrifuge the fermentation broth of Enterobacter holmieae, adjust the solid content of the bacterial broth to 60%, add 10% desiccant to the adjusted bacterial broth to obtain bacterial slurry. The desiccant is galactose: dimethyl sulfoxide: gelatin = 1:1:1. 2) Mix the Enterobacter holmium slurry and excipients obtained in step 1) at a mass ratio of 4:2 and granulate to obtain solid particles. The excipients are wood ash and vermiculite powder, with a wood ash:vermiculite powder ratio of 2:1. 3) The solid particles obtained in step 2) are vacuum dried at a temperature of 30°C for 10 hours to obtain the composite microorganisms.

[0048] Comparative Example 4 The microbial inoculants in this comparative example contain Klebsiella pneumoniae and Enterobacter cholerae, but do not contain humic acid. The fermentation methods for Reiberis and Enterobacter holmie in this comparative example are the same as those in Example 1.

[0049] The preparation method of the composite microorganisms in this comparative example is as follows: 1) Centrifuge the Klebsiella fermentation broth and Enterobacter hominis separately, adjust the solid content of the bacterial broth to 60%, add 10% desiccant to the adjusted bacterial broth to obtain bacterial slurry. The desiccant is galactose: dimethyl sulfoxide: gelatin = 1:1:1. 2) Mix the Klebsiella pneumoniae slurry, Enterobacter holmie slurry, and excipients obtained in step 1) in a mass ratio of 2:2:2 and granulate to obtain solid particles. The excipients are wood ash and vermiculite powder, with a wood ash:vermiculite powder ratio of 2:1. 3) The solid particles obtained in step 2) are vacuum dried at a temperature of 30°C for 10 hours to obtain the composite microorganisms.

[0050] Comparative Example 5 The microbial inoculants in this comparative example contain *Enterobacter holmium* and humic acid, but not *Klebsiella pneumoniae*. Unlike Example 1, this comparative example does not include Klebsiella fermentation.

[0051] The preparation method of the composite microorganisms in this comparative example is as follows: 1) Centrifuge the fermentation broth of Enterobacter holmieae, adjust the solid content of the bacterial broth to 60%, add 10% desiccant to the adjusted bacterial broth to obtain bacterial slurry. The desiccant is galactose: dimethyl sulfoxide: gelatin = 1:1:1. 2) Mix the *Enterobacter holmella* slurry, humic acid, and excipients obtained in step 1) in a mass ratio of 2:2:2 and granulate to obtain solid particles. The excipients are wood ash and vermiculite powder, with a wood ash:vermiculite powder ratio of 2:1. 3) The solid particles obtained in step 2) are vacuum dried at a temperature of 30°C for 10 hours to obtain the composite microorganisms.

[0052] Comparative Example 6 The microbial inoculants in this comparative example contain Klebsiella pneumoniae and humic acid, but do not contain Enterobacter cholerae. Unlike Example 1, this comparative example does not include fermentation with Enterobacter holmieae.

[0053] The preparation method of the composite microorganisms in this comparative example is as follows: 1) Centrifuge the fermentation broth of Enterobacter holmieae, adjust the solid content of the bacterial broth to 60%, add 10% desiccant to the adjusted bacterial broth to obtain bacterial slurry. The desiccant is galactose: dimethyl sulfoxide: gelatin = 1:1:1. 2) Mix the *Enterobacter holmella* slurry, humic acid, and excipients obtained in step 1) in a mass ratio of 2:2:2 and granulate to obtain solid particles. The excipients are wood ash and vermiculite powder, with a wood ash:vermiculite powder ratio of 2:1. 3) The solid particles obtained in step 2) are vacuum dried at a temperature of 30°C for 10 hours to obtain the composite microorganisms.

[0054] Comparative Example 7 This comparative example provides a salt- and alkali-resistant compound microbial agent: Replace *Enterobacter holmie* and *Klebsiella pneumoniae* with heat-resistant phosphate-solubilizing bacteria and *Bacillus subtilis*.

[0055] Experimental Example 1 The composite microbial agents prepared in Examples 1, 2, 3 and Comparative Examples 1-7 were applied to soils with different salinity and compaction levels, respectively. The changes in soil compaction and salinity after one month are shown in Tables 1-3.

[0056] Table 1. Effects of microbial inoculants on the degree of compaction in mildly saline-alkali soil.

[0057] Table 2. Effects of microbial inoculants on the degree of compaction in moderately saline-alkali soil.

[0058] Table 3. Effects of microbial inoculants on the degree of compaction in severely saline-alkali soil.

[0059] As shown in Tables 1-3, the composite microbial agent prepared by this invention has a good effect on different degrees of saline-alkali soil compaction. However, the microbial species that have not been acclimatized by the specific pH gradient of this invention have a poor effect on saline-alkali soil compaction. The single microbial species has a poor effect on saline-alkali soil compaction. Humic acid not only acts as a carrier and binder, but it can also improve soil structure, enhance water and fertilizer retention capacity, stimulate microbial activity, and synergistically promote the formation and stability of soil aggregates with the microbial cells.

[0060] Experiment Example 2 Example 1, Comparative Examples 2-7, and humic acid were applied to saline-alkali soils used for planting corn. The changes in soil compaction, salinity, and corn yield after 4 months are as follows: Table 4. Effects of microbial inoculants on maize cultivation in saline-alkali soil

[0061] As shown in Table 4, the compound microbial agent prepared by this invention can further increase crop yield after repairing soil compaction.

[0062] Experimental Example 3 The changes in the effective viable count of the compound microbial agent obtained in Implementation 1 when stored at room temperature are shown in Table 5.

[0063] Table 5

[0064] As shown in Table 5, the microbial agent of the present invention has good stability and remains effective for 24 months.

[0065] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A salt- and alkali-resistant compound microbial agent, characterized in that, The microbial agent includes Klebsiella pneumoniae, Enterobacter holmium, and humic acid, with the ratio of Klebsiella pneumoniae: Enterobacter holmium: Humic acid = 1:0.5~2:1~3. Klebsiella pneumoniae and Enterobacter holmium are obtained by fermentation culture.

2. The salt- and alkali-resistant compound microbial agent according to claim 1, characterized in that, The fermentation culture method of Klebsiella includes: firstly, controlling the reaction conditions to conduct a preliminary culture of Klebsiella to obtain a primary seed culture; then, conducting a secondary culture of the primary seed culture to obtain a secondary seed culture; finally, conducting a staged fermentation of the secondary seed culture under different pH gradients to obtain Klebsiella fermentation broth; wherein the pH gradient is 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, 9.5~10.0, and the culture time for each stage is 5~10 hours.

3. The salt- and alkali-resistant compound microbial agent according to claim 2, characterized in that, The initial culture conditions are as follows: shake culture in an incubator at a temperature of 25~40℃ for 18~30h, with the rotation speed controlled at 150~200rpm.

4. The salt- and alkali-resistant compound microbial agent according to claim 2, characterized in that, The secondary culture conditions are: temperature 25~40℃, rotation speed 150~200rpm, pH 6.5~7.5, dissolved oxygen 20-50%, and culture time 18~30h.

5. The salt- and alkali-resistant compound microbial agent according to claim 2, characterized in that, The sodium chloride concentrations corresponding to the pH gradients are 8~15 g / L, 15~30 g / L, 30~50 g / L, 50~80 g / L, and 80~100 g / L.

6. The salt- and alkali-resistant compound microbial agent according to claim 1, characterized in that, The fermentation culture method of Enterobacter holmie includes: firstly, controlling the reaction conditions to perform a preliminary culture of Enterobacter holmie to obtain a primary seed culture; then, performing a secondary culture of the primary seed culture to obtain a secondary seed culture; finally, performing a staged fermentation of the secondary seed culture under different pH gradients to obtain Enterobacter holmie fermentation broth; wherein the pH gradient is 7.2~8.0, 8.0~8.5, 8.5~9.0, 9.0~9.5, 9.5~10.0, and the culture time for each stage is 5~10 hours.

7. The salt- and alkali-resistant composite microbial agent according to claim 6, characterized in that, The initial culture conditions are as follows: shake culture in an incubator at a temperature of 25~40℃ for 18~30h, with the rotation speed controlled at 150~200rpm.

8. The salt- and alkali-resistant compound microbial agent according to claim 6, characterized in that, The secondary culture conditions are: temperature 25~40℃, rotation speed 150~200rpm, dissolved oxygen 20-50%, and culture time 18~30h.

9. The salt- and alkali-resistant composite microbial agent according to claim 6, characterized in that, The sodium chloride concentrations corresponding to the pH gradients are 8~15 g / L, 15~30 g / L, 30~50 g / L, 50~80 g / L, and 80~100 g / L.

10. The method for preparing the salt- and alkali-resistant composite microbial agent according to any one of claims 1-9, characterized in that, The method includes: Step 1: Centrifuge the Klebsiella fermentation broth and the Enterobacter hopnea fermentation broth separately, adjust the solid content of the bacterial broth to 50-80%, and add 0.5-15% of the desiccant added to the bacterial broth to obtain Klebsiella bacterial slurry and Enterobacter hopnea bacterial slurry, respectively. Step 2: Mix the Klebsiella pneumoniae slurry, Enterobacter holmium slurry, humic acid and excipients from Step 1 in a certain proportion and granulate to obtain solid particles; Step 3: Vacuum dry the solid particles from Step 2 to obtain the composite microbial agent.

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