A high-salt-tolerance high-content composite microbial agent based on a double-system saline hydroponic medium and a preparation method thereof

Abstract

The application provides a kind of high content composite microbial inoculant based on double system saline-alkali water culture medium and its preparation method, the composite microbial inoculant is compounded by A system fermentation broth and B system fermentation broth according to the volume ratio 4:1;A system fermentation broth is salt stress resistant EM microbial population fermentation broth, salt stress resistant EM microbial population is composed of Rhodopseudomonas palustris, Lactobacillus plantarum and Saccharomyces cerevisiae, the proportion of viable bacterial count is (30%-40%): (35%-45%): (20%-30%) in turn, the total number of viable bacteria in A system fermentation broth is greater than or equal to 100 billion per milliliter;B system fermentation broth is nitrifying functional microbial population fermentation broth, nitrifying functional microbial population is composed of Nitrosomonas and Nitrobacter, the proportion of viable bacterial count is (50%-60%): (40%-50%), the total number of viable bacteria in B system fermentation broth is greater than or equal to 20 billion per milliliter;The total number of viable bacteria of composite microbial inoculant is greater than or equal to 100 billion per milliliter. The high content composite microbial inoculant based on double system saline-alkali water culture medium and its preparation method can solve the problems of salt resistance and nutrition in a synergistic manner.

Description

Technical Field

[0001] This invention relates to the field of microbial and soil improvement technology, and more specifically, to a high-content compound microbial agent for salt stress resistance based on a dual-system saline-alkali water culture medium and its preparation method. Background Technology

[0002] Soil salinization causes severe salt stress, significantly limiting crop growth and triggering a series of physiological problems such as osmotic stress, ion toxicity, and nutrient imbalance. Microbial agents, such as EM (Effective Microbial Community), are considered a green solution. However, existing technologies face the following bottlenecks: 1. The single function of microbial agents is not compatible with the environment. Ordinary EM microbial agents focus on improving the micro-ecology, but lack the function to address the core pain point of "severe nitrogen volatilization loss and extremely low fertilizer utilization rate" in saline-alkali land. At the same time, the microbial agents are prepared in freshwater environment and have a low survival rate after being applied to saline-alkali land. 2. Antagonism between bacterial species prevents efficient compounding. Powerful nitrifying bacteria can convert ammonium nitrogen, which is difficult for crops to utilize, into easily absorbed nitrate nitrogen, greatly improving the nitrogen fertilizer utilization rate in saline-alkali land. However, there is a fundamental conflict between nitrifying bacteria (aerobic, neutral pH-loving) and EM core bacteria such as lactic acid bacteria (anaerobic, acid-producing) in terms of fermentation conditions, making it impossible to obtain compound products with high viable counts through simple mixed fermentation. 3. Low live bacteria content and insignificant effect: Commercially available bacterial agents generally have low live bacteria counts (often below 1 billion / ml), and cannot form an effective bacterial community advantage in harsh saline-alkali soils.

[0003] Therefore, there is an urgent need in this field for a high-content compound microbial agent that can synergistically solve the dual problems of salt resistance and nutrition, and for all strains to be pre-acclimatized in a saline-alkali environment, and to break through the technical barriers to its industrial preparation. Summary of the Invention

[0004] The present invention aims to solve the technical problems mentioned in the background art and provide a high-content compound microbial agent for salt stress resistance based on a dual-system saline-alkali water culture medium and its preparation method. The primary objective is to provide a compound microbial agent that can both enhance crop salt tolerance and improve nitrogen fertilizer utilization. Another objective is to provide a dedicated dual-system saline-alkali water culture medium for the microbial agent and its preparation method to resolve the fermentation contradictions among functional microbial groups. The ultimate objective is to provide the application of this microbial agent in alleviating crop salt stress and promoting crop growth in saline-alkali land.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a high-content compound microbial agent for salt stress resistance based on a dual-system saline-alkali water culture medium, wherein the compound microbial agent is prepared by mixing fermentation broth of system A and fermentation broth of system B in a volume ratio of 4:1; The fermentation broth of System A is a salt-stress-resistant EM bacterial culture fermentation broth. The salt-stress-resistant EM bacterial culture is composed of Rhodopseudomonas palustris, Lactobacillus plantarum and Saccharomyces cerevisiae, with the live bacteria count ratios being (30%~40%): (35%~45%): (20%~30%), and the total live bacteria count of System A fermentation broth is ≥10 billion / mL. The fermentation broth of System B is a fermentation broth of nitrifying functional bacteria, which consists of *Nitrostomyces* and *Nitrobacterium*, with a live bacteria ratio of (50%~60%): (40%~50%). The total number of live bacteria in the fermentation broth of System B is ≥2 billion / ml. The total number of live bacteria in the compound microbial agent is ≥10 billion / ml.

[0006] A dual-system saline-alkali water culture medium for preparing the above-mentioned compound bacterial agent includes a special high-density culture medium for system A and a special culture medium for system B, wherein both the special high-density culture medium for system A and the special culture medium for system B are based on saline-alkali water with a salinity of 1.6‰~2.0‰ and a pH of 8.5~9.0; The saline water is natural well water from southern Xinjiang. The high-density culture medium for System A, per 1L of saline-alkali water, comprises the following components and contents: glucose 35g~50g, glycerol 5g~10g, yeast extract 20g~30g, peptone 5g~10g, potassium dihydrogen phosphate 3g~5g, magnesium sulfate 1.5g~2.5g, manganese sulfate 0.05g~0.15g, L-cysteine ​​hydrochloride 0.2g~0.4g, and Tween-80 1mL~2mL; The B system-specific culture medium, per 1L of saline-alkali water, consists of the following components and contents: sodium bicarbonate 1.0g~2.0g, ammonium sulfate 1.0g~2.0g, potassium dihydrogen phosphate 0.5g~1.0g, magnesium chloride 0.1g~0.3g, calcium chloride 0.01g~0.05g, and trace element solution 1mL~2mL.

[0007] A method for preparing a high-content compound bacterial agent for salt stress resistance based on the above-described dual-system saline-alkali water culture medium includes the following steps: (1) Fermentation of System A: Rhodopseudomonas palustris, Lactobacillus plantarum and Saccharomyces cerevisiae are mixed at a live cell ratio of (30%~40%): (35%~45%): (20%~30%) and inoculated into the dedicated high-density culture medium of System A at an inoculation rate of 4%~6%; under anaerobic / facultative anaerobic conditions at 35-37℃, the pH is controlled at 5.8-6.2 by automatic addition of alkali solution, and fermentation is carried out for 48-60 hours using a fed-batch strategy to obtain fermentation broth of System A; (2) Fermentation of System B: Mix Bacillus nitrite and Bacillus nitrification at a viable count ratio of (50%~60%): (40%~50%), and inoculate them into the special culture medium for System B at an inoculation rate of 5%~10%; under strong aeration and aerobic conditions at 25-30℃, control the pH at 7.0-7.5, and ferment for 5-7 days to obtain fermentation broth of System B; (3) Compound preparation: After the fermentation of systems A and B is completed, the number of viable bacteria in the fermentation broth of system A and system B is tested respectively, and they are mixed at a volume ratio of 4:1 to prepare the final compound microbial agent.

[0008] The beneficial effects of this invention are as follows: This invention successfully integrates salt-stress-resistant EM bacteria and highly efficient nitrifying bacteria into a single product through independent dual-system fermentation and physical compounding. This overcomes the antagonistic limitations between bacterial species, achieving functional synergy between biological stress resistance and biological fertilizer efficiency enhancement. It simultaneously solves the dual problems of poor crop growth (salt stress) and insufficient nitrogen fertilizer utilization in saline-alkali land. All bacterial strains in systems A and B are domesticated and cultivated in a saline-alkali water culture medium based on natural well water from southern Xinjiang. They have adapted to the high-salt, high-pH environment of saline-alkali land. After application to the soil, their survival rate and colonization capacity far exceed those of traditional freshwater-prepared bacterial agents, allowing for rapid formation of dominant bacterial communities and stable improvement effects. By independently optimizing the fermentation process and culture medium formula for each system, each bacterial community reaches optimal growth conditions. The compound microbial agent achieves a high-density growth rate, with a final total viable count of ≥10 billion / mL. Sufficient functional bacteria introduced into the field effectively resist the inhibitory effects of the saline-alkali environment, ensuring stable and significant improvement results. This invention uses abundant natural saline-alkali water from southern Xinjiang as its core production raw material, transforming a limited resource into a useful one, turning waste into treasure, and significantly reducing the cost of culture medium preparation. Simultaneously, the industrial fermentation process is simple and controllable, easy to scale up, and highly valuable for promotion. Field trials have confirmed that the compound microbial agent of this invention can increase the seedling emergence rate of crops in saline-alkali land by more than 25%, and prevents stunted growth during the mid-growth stage of crops; it also increases nitrogen fertilizer utilization by more than 20%, reducing nitrogen volatilization loss; ultimately, it can increase crop yield by 15%-35%, significantly improving the economic benefits of agricultural production in saline-alkali land. Attached Figure Description

[0009] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are only drawings of some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings. In addition, the drawings in the following description can be regarded as schematic diagrams and are not intended to limit the actual size of the product, the actual flow of the method, the actual timing of the signals, etc. involved in the embodiments of the present invention.

[0010] Figure 1This is a schematic diagram of the preparation method steps of the present invention. Detailed Implementation

[0011] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0012] like Figure 1 As shown in the figure, the present invention provides a high-content compound bacterial agent for salt stress resistance based on a dual-system saline-alkali water culture medium. The compound bacterial agent is composed of fermentation broth from system A and fermentation broth from system B in a volume ratio of 4:1. System A fermentation broth is a salt-stress-resistant EM (Effective Microorganisms) fermentation broth. The salt-stress-resistant EM community consists of Rhodopseudomonas palustris, Lactobacillus plantarum, and Saccharomyces cerevisiae, with the viable cell count ratios being (30%~40%): (35%~45%): (20%~30%), and the total viable cell count of System A fermentation broth is ≥10 billion / mL. Among them, Rhodopseudomonas palustris is a photosynthetic strain that can use light energy to fix carbon dioxide, produce a variety of growth-promoting substances, and enhance crop stress resistance; Lactobacillus plantarum can produce organic acids to regulate the microenvironment, inhibit the growth of harmful bacteria, and improve the physical and chemical properties of the soil; Saccharomyces cerevisiae can secrete extracellular polysaccharides, enhance the soil's water and fertilizer retention capacity, and provide nutritional support for the growth of other microbial species.

[0013] The fermentation broth of System B is a fermentation broth containing nitrifying functional bacteria. The nitrifying functional bacteria consist of *Nitrostomyces* and *Nitrobacterium*, with a viable count ratio of (50%~60%): (40%~50%). The total number of viable bacteria in the fermentation broth of System B is ≥2 billion / mL. Among them, *Nitrostomyces* can convert ammonium nitrogen into nitrite nitrogen, and *Nitrobacterium* can further convert nitrite nitrogen into nitrate nitrogen. The two work synergistically to achieve a highly efficient nitrification process, significantly improving nitrogen fertilizer utilization.

[0014] After compounding, the final compound microbial agent has a total live bacteria count of ≥10 billion / ml, and can simultaneously possess the dual functions of salt stress resistance and high-efficiency nitrification.

[0015] A dual-system saline-alkali water culture medium for preparing the above-mentioned compound bacterial agent includes a special high-density culture medium for system A and a special culture medium for system B. Both the special high-density culture medium for system A and the special culture medium for system B are based on saline-alkali water with a salinity of 1.6‰~2.0‰ and a pH of 8.5~9.0. The saline-alkali water is natural well water from southern Xinjiang, enabling the simultaneous in-situ acclimatization of all bacterial species and significantly improving their adaptability to the saline-alkali environment of southern Xinjiang. The high-density culture medium for System A, per 1L of saline-alkali water, comprises the following components and contents: glucose 35g~50g, glycerol 5g~10g, yeast extract 20g~30g, peptone 5g~10g, potassium dihydrogen phosphate 3g~5g, magnesium sulfate 1.5g~2.5g, manganese sulfate 0.05g~0.15g, L-cysteine ​​hydrochloride 0.2g~0.4g, and Tween-80 1mL~2mL. Glucose and glycerol serve as carbon sources, providing energy for the System A microbial community; yeast extract and peptone act as nitrogen sources and growth factors, promoting rapid microbial reproduction; potassium dihydrogen phosphate, magnesium sulfate, and manganese sulfate provide mineral elements to meet the physiological metabolic needs of the microorganisms; L-cysteine ​​hydrochloride acts as a reducing agent, maintaining the stability of the anaerobic / facultative anaerobic fermentation environment; and Tween-80 acts as a surfactant, increasing the solubility of lipid-soluble substances, promoting cell membrane permeability, and enhancing the high-density fermentation effect.

[0016] The B system's dedicated culture medium, per 1L of saline-alkali water, comprises the following components and contents: sodium bicarbonate 1.0g~2.0g, ammonium sulfate 1.0g~2.0g, potassium dihydrogen phosphate 0.5g~1.0g, magnesium chloride 0.1g~0.3g, calcium chloride 0.01g~0.05g, and trace element solution 1mL~2mL. Among these, ammonium sulfate serves as the core nitrogen source, providing substrates for nitrification by nitrifying bacteria; sodium bicarbonate adjusts the pH of the culture medium, maintaining a slightly alkaline growth environment suitable for the growth requirements of nitrifying bacteria; potassium dihydrogen phosphate, magnesium chloride, calcium chloride, and trace element solution provide essential mineral nutrients for nitrifying bacteria, ensuring their efficient growth and metabolism.

[0017] A method for preparing a high-content compound bacterial agent resistant to salt stress based on the above-mentioned dual-system saline-alkali water culture medium includes the following steps: (1) Fermentation of System A: Rhodopseudomonas palustris, Lactobacillus plantarum and Saccharomyces cerevisiae are mixed at a viable count ratio of (30%~40%): (35%~45%): (20%~30%) and inoculated into a dedicated high-density culture medium for System A at an inoculation rate of 4%~6%. Under anaerobic / facultative anaerobic conditions at a temperature of 35-37℃, the pH is controlled at 5.8-6.2 by automatically adding alkali solution during fermentation. The fermentation is carried out for 48-60 hours using a fed-batch strategy to obtain fermentation broth for System A. After fermentation, the viable count is tested to ensure that the total number of viable bacteria in the fermentation broth for System A is ≥10 billion / mL. In System A, Bacillus spp. (which has the functions of potassium solubilization and extracellular polysaccharide production) can be added as a functional supplement, but its content shall not exceed 10% of the total number of bacteria in System A to avoid affecting the synergistic effect of the original bacterial community. (2) Fermentation of System B: Mix *Bacillus nitrosatus* and *Bacillus nitrosatus* at a viable count ratio of (50%~60%): (40%~50%), and inoculate them into the special culture medium for System B at an inoculation rate of 5%~10%; under the conditions of 25-30℃ and strong aeration and aerobic conditions, control the pH at 7.0-7.5, and ferment for 5-7 days to obtain the fermentation broth of System B. After the fermentation is completed, test the viable count to ensure that the total number of viable bacteria in the fermentation broth of System B is ≥2 billion / mL; in System B, other nitrifying bacteria (such as *Typha nitrosatus*) that have been screened for saline-alkali environments can be used to replace *Bacillus nitrosatus* or *Bacillus nitrosatus*, as long as they can achieve efficient nitrification function; (3) Compound preparation: After the fermentation of systems A and B is completed, the number of viable bacteria in the fermentation broth of system A and system B is tested respectively, and they are mixed at a volume ratio of 4:1 to prepare the final compound microbial agent. In addition, according to the growth requirements of specific crops (such as cotton, wheat, corn, etc.) and the salinity level of saline-alkali land, the volume ratio of the compound preparation of system A and system B fermentation broth can be adjusted between 3:1 and 5:1 to ensure that the best salt resistance and fertilizer efficiency can be achieved in different scenarios.

[0018] The aforementioned liquid compound microbial agent can be further adsorbed onto carriers such as peat moss and wheat bran to form a solid microbial agent, extending its shelf life and facilitating transportation; it can also be mixed with stress-resistance adjuvants such as humic acid and seaweed extract to form a high-end functional liquid fertilizer, further enhancing its salt stress resistance effect.

[0019] This invention's compound microbial agent can be widely applied to crop cultivation improvement in saline-alkali lands such as southern Xinjiang and northwestern inland areas, especially suitable for major crops such as cotton, wheat, corn, and tomatoes. Specific application methods include: seed treatment before sowing, root irrigation during the seedling stage, foliar spraying during the growing season, or drip irrigation. It can effectively alleviate salt stress in crops, promote root growth and plant development, improve nitrogen fertilizer utilization, and increase crop yield. Simultaneously, this microbial agent can also be used in saline-alkali land soil improvement projects to improve the soil microecological environment, reduce soil salinity, and enhance soil fertility.

[0020] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A high-content compound bacterial agent for salt stress resistance based on a dual-system saline-alkali water culture medium, characterized in that, The compound microbial agent is prepared by mixing fermentation broth from system A and fermentation broth from system B at a volume ratio of 4:

1. The fermentation broth of System A is a salt-stress-resistant EM bacterial culture fermentation broth. The salt-stress-resistant EM bacterial culture is composed of Rhodopseudomonas palustris, Lactobacillus plantarum and Saccharomyces cerevisiae, with the live bacteria count ratios being (30%~40%): (35%~45%): (20%~30%), and the total live bacteria count of System A fermentation broth is ≥10 billion / mL. The fermentation broth of System B is a fermentation broth of nitrifying functional bacteria, which consists of *Nitrostomyces* and *Nitrobacterium*, with a live bacteria ratio of (50%~60%): (40%~50%). The total number of live bacteria in the fermentation broth of System B is ≥2 billion / ml. The total number of live bacteria in the compound microbial agent is ≥10 billion / ml.

2. A dual-system saline-alkali water culture medium for preparing the composite bacterial agent of claim 1, characterized in that, It includes a high-density culture medium specifically for System A and a culture medium specifically for System B, both of which are based on saline-alkaline water with a salinity of 1.6‰ to 2.0‰ and a pH of 8.5 to 9.

0. The saline water is natural well water from southern Xinjiang. The high-density culture medium for System A, per 1L of saline-alkali water, comprises the following components and contents: glucose 35g~50g, glycerol 5g~10g, yeast extract 20g~30g, peptone 5g~10g, potassium dihydrogen phosphate 3g~5g, magnesium sulfate 1.5g~2.5g, manganese sulfate 0.05g~0.15g, L-cysteine ​​hydrochloride 0.2g~0.4g, and Tween-80 1mL~2mL; The B system-specific culture medium, per 1L of saline-alkali water, consists of the following components and contents: sodium bicarbonate 1.0g~2.0g, ammonium sulfate 1.0g~2.0g, potassium dihydrogen phosphate 0.5g~1.0g, magnesium chloride 0.1g~0.3g, calcium chloride 0.01g~0.05g, and trace element solution 1mL~2mL.

3. A method for preparing a high-content composite bacterial agent for salt stress resistance based on the dual-system saline-alkali water culture medium according to claim 2, characterized in that, Includes the following steps: (1) Fermentation of System A: Rhodopseudomonas palustris, Lactobacillus plantarum and Saccharomyces cerevisiae are mixed at a live cell ratio of (30%~40%): (35%~45%): (20%~30%) and inoculated into the dedicated high-density culture medium of System A at an inoculation rate of 4%~6%; under anaerobic / facultative anaerobic conditions at 35-37℃, the pH is controlled at 5.8-6.2 by automatic addition of alkali solution, and fermentation is carried out for 48-60 hours using a fed-batch strategy to obtain fermentation broth of System A; (2) Fermentation of System B: Mix Bacillus nitrite and Bacillus nitrification at a viable count ratio of (50%~60%): (40%~50%), and inoculate them into the special culture medium for System B at an inoculation rate of 5%~10%; under strong aeration and aerobic conditions at 25-30℃, control the pH at 7.0-7.5, and ferment for 5-7 days to obtain fermentation broth of System B; (3) Compound preparation: After the fermentation of systems A and B is completed, the number of viable bacteria in the fermentation broth of system A and system B is tested respectively, and they are mixed at a volume ratio of 4:1 to prepare the final compound microbial agent.

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