Complex microbial inoculant for repairing saline-alkali soil and application of complex microbial inoculant

Abstract

The invention discloses a complex microbial inoculant for repairing saline-alkali soil and application of the complex microbial inoculant, and belongs to the technical field of microorganisms. The preparation method comprises the following steps: respectively carrying out fermentation culture on sphingobacterium bambusae and deinococcus cellulosilyticus, carrying out centrifugal resuspension, and mixing the obtained bacterial solutions, so as to prepare the composite bacterial agent for repairing the soil of the saline-alkali land. The preparation method comprises the following steps: carrying out fermentation culture on the sphingobacterium bambusae and the deinococcus cellulosilyticus respectively, and carrying out centrifugal resuspension on the obtained bacterial solutions, so as to obtain the composite bacterial agent for repairing the soil of the saline-alkali land. According to the invention, the complex microbial inoculant is applied to saline-alkali soil by using crushed corn straw as a carrier; experimental results show that the complex microbial inoculant can efficiently reduce the pH value and salt content of the saline-alkali soil and improve the organic matter content of the saline-alkali soil. A novel microbial material and an efficient remediation method are provided for improvement and remediation of the saline-alkali soil, the steps are simple, the effect is lasting and stable, and the method has prominent practical application value and wide application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a compound microbial agent for remediating saline-alkali soil and its application. Background Technology

[0002] Soil salinization refers to the process by which soil contains excessive amounts of soluble salts (mainly sulfates, chlorides, carbonates, and bicarbonates of metallic elements such as sodium, potassium, calcium, and magnesium) and alkaline substances due to scarce rainfall, intense evaporation, or other factors, leading to the deterioration of soil's physical and chemical properties and the formation of saline-alkali land. Soil salinization severely restricts crop growth, resulting in reduced yields per unit area and even crop failure. It adversely affects the development of agriculture, forestry, and animal husbandry. Therefore, how to restore saline-alkali soil, curb salinization, expand arable land, and improve the ecological environment is an urgent problem to be solved.

[0003] Traditional physical or chemical remediation techniques for improving saline-alkali soils have limitations such as high cost and susceptibility to secondary pollution. In contrast, microbial remediation technology improves soil structure and regulates saline-alkali stress through microbial metabolism, increases the availability of nutrients in the soil, promotes plant absorption of nutrients and water, and enhances plant resistance. There are many ways to improve saline-alkali soils with microorganisms. Common methods include producing microbial fertilizers or microbial preparations, which can be applied directly to the soil or used in combination with other fertilizers. Microbial fertilizers have advantages such as good fertilization efficiency, zero environmental pollution, maintenance of soil ecosystem balance, and enrichment of soil fertility, making them an important part of agricultural production. In recent years, significant progress has been made in the screening of functional microbial strains for saline-alkali soil remediation, the construction of synthetic microbial communities, and the combined use of multiple technologies. However, technical bottlenecks still exist in key areas such as the environmental adaptability of microbial strains, the sustainability and stability of remediation effects, the impact of pollutants on the effectiveness of microbial remediation, and the large-scale commercialization of microbial remediation technology.

[0004] Therefore, there is an urgent need to provide a microbial agent that is highly adaptable to the environment, has a long-lasting and stable remediation effect, and is not easily affected by pollutants, so as to effectively remediate saline-alkali soil and reverse soil salinization. Summary of the Invention

[0005] The purpose of this invention is to provide a compound microbial agent for remediating saline-alkali soil and its application, in order to solve the problems existing in the prior art. The compound microbial agent provided by this invention can effectively reduce the pH value and salt content of saline-alkali soil, and increase the organic matter content of saline-alkali soil. It is used to improve saline-alkali soil and has the advantages of simple steps and stable effects.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] The present invention provides a microbial composition for remediating saline-alkali soil, comprising Sphingobacterium bambusae and Deinococcus cellulosilyticus.

[0008] The present invention also provides an application of the above-mentioned microbial composition in the preparation of a compound microbial agent for remediating saline-alkali soil.

[0009] This invention also provides a method for preparing a compound microbial agent for remediating saline-alkali soil, comprising the following steps:

[0010] After activating the above-mentioned *Sphingomonas sphingolipidae* and *Coccidioidomyces fibronectin*, they were fermented, centrifuged, the precipitate was collected and resuspended to obtain a bacterial solution; the bacterial solution was mixed to obtain the compound bacterial agent.

[0011] Furthermore, the ratio of viable bacteria of *Sphingomonas sphingolipidae* to viable bacteria of *Coccidioidomyces fibronectin* is 1:1.

[0012] Furthermore, in the compound microbial agent, the total viable count of *Sphingomyelin-Bacillus basilata* and *Tectus fibrolyticus* is not less than 1 × 10⁻⁶. 8 CFU / mL.

[0013] The present invention also provides a compound microbial agent obtained according to the above preparation method.

[0014] The present invention also provides the application of the above-mentioned microbial composition or the above-mentioned compound microbial agent in the remediation of saline-alkali soil.

[0015] The present invention also provides a method for remediating saline-alkali soil, comprising the following steps:

[0016] Crush the corn stalks and apply them evenly to the saline-alkali soil. Spray the compound microbial agent mentioned above evenly onto the surface of the crushed corn stalks, and then plow the soil.

[0017] Furthermore, the volume-to-mass ratio of the compound microbial agent to the corn stalk is 1L:4kg.

[0018] Furthermore, the application rate of corn stalks is 3600 kg / hm². 2 .

[0019] The present invention discloses the following technical effects:

[0020] This invention involves the separate fermentation and centrifugation of *Sphingobacterium bambusae* and *Deinococcus cellulosilyticus*, followed by resuspension, to create a composite microbial agent for remediating saline-alkali soils. This agent is applied to saline-alkali soils using pulverized corn stalks as a carrier. Experimental results show that this composite agent can effectively reduce the pH and salinity of saline-alkali soils while increasing their organic matter content. This invention provides a novel microbial material and an efficient remediation method for improving and restoring saline-alkali soils. The process is simple, the effects are long-lasting and stable, and it has significant practical application value and broad application prospects. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Existing technologies for remediating saline-alkali soil using microbial agents include: a composite soil remediation material, its preparation method, and its application. This composite soil remediation material is loaded with 15-25 parts of Scheres' mineral, 5-10 parts of biosurfactant, 10-20 parts of edible mushroom residue, 20-30 parts of natural minerals, 1-5 parts of bicarbonate, and 70-80 parts of water, all contained in a microbial inoculum. The microbial inoculum includes *Arthrobacter spheroidae*, *Serratia marcescens*, and *Pichia pastoris*. *Arthrobacter spheroidae* cells possess a certain osmotic pressure regulation mechanism. In high-salt environments, *Arthrobacter spheroidae* can absorb salt from the soil into its cells, reducing the salt content in the soil. It then utilizes its metabolic activities to mutually sublimate the absorbed salt ions, thereby reducing the alkalinity of the soil. *Serratia marcescens* cells avoid carrying a charge and can adsorb harmful salt ions such as sodium ions from the soil through ion exchange, fixing the salt ions on the cell surface and reducing their concentration in the soil. Furthermore, *Serratia marcescens* can improve soil structure, increase soil porosity, and allow salts to be expelled from the soil with water flow. During its metabolism, it produces acidic substances that can neutralize soil alkalinity and lower the soil pH. *Pichia pastoris* produces organic acids during its growth and metabolism, which can neutralize alkaline components in the soil and reduce soil salinization. Moreover, *Pichia pastoris* can activate soil salts, decompose organic matter in the soil, release nutrients needed for plant growth, and secrete plant growth hormones, stimulating root growth and development and improving plant tolerance to saline-alkali environments.

[0027] Existing technologies for using microbial agents to remediate saline-alkali soils include: a salt-tolerant compound microbial agent comprising the following components in parts by weight: 20-50 parts of Bacillus subtilis, 20-60 parts of Bacillus cereus, 10-40 parts of Bacillus amyloliquefaciens, 20-40 parts of Bacillus megaterium, 10-30 parts of Paracoccus denitrifyingis, 15-30 parts of Lactobacillus buchneri, and 20-40 parts of Candida albicans. This compound microbial agent is prepared by a mixed symbiotic culture method of multiple microbial strains. During the culture process, the integrity of the microbial degradation chain is maintained, and the diversity of functional microorganisms is ensured, making it easier to adapt to different saline-alkali soils. The compound microbial agent contains a high number of viable bacteria, thus containing highly active metabolites. After being applied to saline-alkali soils, it can effectively increase the microbial community in the soil, rapidly decompose organic matter for plant absorption and utilization, and the produced organic acids can effectively neutralize the alkali in the soil, thereby effectively reducing soil alkalinity and enhancing soil permeability, water retention, and aeration.

[0028] Existing technologies for using microbial agents to remediate saline-alkali soil include: a composite soil conditioner for saline-alkali soil, prepared from the following raw materials: biodegradable microspheres containing microorganisms, straw powder, furfural residue, diatomaceous earth, and mixed grass powder; the mixed grass powder is prepared by mixing and grinding *Curculigo orchioides*, *Phragmites australis*, and *Festuca pulveratum* in a mass ratio of (5-10):(2-5):2; the preparation method of the biodegradable microspheres containing microorganisms is as follows: S1. Preparation of the composite microbial preparation: Klebsiella pneumoniae, *Pseudomonas aeruginosa*, *Rhodospirillum*, and *Pseudomonas* are dispersed in sterile water to prepare 10 7 S2. Preparation of aqueous phase: Sodium acrylate and acrylamide are dissolved in water, surfactant is added, and the mixture is stirred evenly to obtain an aqueous phase; S3. Preparation of biodegradable microspheres containing microorganisms: The compound bacterial preparation in step S1 is added to the aqueous phase in step S2, initiator and crosslinking agent are added, the mixture is stirred evenly, emulsified, filtered, and dried to obtain biodegradable microspheres containing microorganisms.

[0029] Existing technologies for using microbial agents to remediate saline-alkali soil include: a microbial agent for improving saline-alkali soil, comprising the following components: 8-10 parts of an organic mixture adsorbed with a compound microbial liquid agent, 40-60 parts of decomposed organic matter, 3-6 parts of Bacillus magaterium agent, 25-40 parts of Bacillus mucilaginosus agent, 1-3 parts of Lactobacillus plantarum agent, and 1-3 parts of Candida utilis agent.

[0030] Existing technologies for using microbial agents to remediate saline-alkali soil include: a compound microbial agent specifically for saline-alkali soil, comprising rhizobium YIC4027 and arbuscular mycorrhizal fungus Glomus mosseae (GM). The agent consists of fermentation products or metabolites obtained from either rhizobium YIC4027 or GM, with each strain's fermentation products or metabolites mixed separately with a carrier to obtain solid substances, which are then mixed at a 1:1 mass ratio. The rhizobium YIC4027 and GM are cultured using conventional methods, wherein the rhizobium YIC4027 agent contains approximately 2 × 10⁻⁶ viable bacteria. 7 The number of spores in the GM inoculum of arbuscular mycorrhizal fungi is approximately 20 per gram.

[0031] Existing technologies for using microbial agents to remediate saline-alkali soil include: a composite soil remediation agent suitable for saline-alkali soil, with the following raw material weight ratio: 20-200 parts of composite microbial agent, 80-300 parts of modified potassium humate, 20-50 parts of polyacrylamide, 20-100 parts of chitosan, 50-200 parts of polyaspartic acid, and 600-900 parts of other organic fertilizers; the various raw materials are uniformly mixed by a mixing and stirring mechanism. The compound microbial agent is mainly composed of fermentation broth from 11 microbial strains, including Stenotrophomonas rhizophila X1, Bacillus thuringiensis SMs13, Bacillus licheniformis SMrs15, Bacillus laterosporus LEs15, Bacillus amyloliquefaciens LEr08, Bacillus megaterium FBs03, Bacillus subtilis FBrs14, Bacillus sp. Ds18, Pseudomonas mendocina L1, Pseudomonas stutzeri Dst11, and Lactococcus lactis subsp. lactis.

[0032] The *Sphingobacterium bambusae* strain of this invention has been deposited at the China Center for Type Culture Collection (CCTCCAB) with accession number CCTCCB 209162T, located at Wuhan University, Wuhan, China, on September 8, 2012. The *Deinococcus cellulosilyticus* strain has also been deposited at the same collection center with accession number CCTCCB 207224T, located at Wuhan University, Wuhan, China, on April 13, 2011. The public can purchase these strains from the collection centers.

[0033] The formulation of the R2A agar medium of the present invention is as follows: 0.5g yeast extract, 0.5g peptone, 0.5g casein hydrolysate, 0.5g glucose, 0.5g soluble starch, 0.3g dipotassium hydrogen phosphate, 0.024g anhydrous magnesium sulfate, 0.3g sodium pyruvate, and 15.0g agar, dissolved in 1L sterile water, with a pH of 7.2±0.2.

[0034] Example 1

[0035] *Synthia spp.* was inoculated onto streptomycin-containing agar medium (China Center for Type Culture Collection, catalog number 0113), activated at 31°C for 1 day, then inoculated onto LB medium and fermented at 31°C until OD reached. 600 >3. Centrifuge the fermentation broth at 3000 rpm for 15 min, collect the precipitate, and resuspend it in LB medium until the viable cell count is 1×10⁻⁶. 8 CFU / mL, collect bacterial suspension;

[0036] *Mirabilis fibronectin* was inoculated onto R2A agar medium and activated at 30°C for 1 day. It was then inoculated onto LB medium and fermented at 30°C until OD reached. 600 >3. Centrifuge the fermentation broth at 3000 rpm for 15 min, collect the precipitate, and resuspend it in LB medium until the viable cell count is 1×10⁻⁶. 8 CFU / mL, collect bacterial suspension;

[0037] The bacterial solutions of *Bacillus sphingolipidae* and *Coccus pyogenes* were mixed in equal volumes to form a compound bacterial agent for remediating saline-alkali soil.

[0038] Example 2

[0039] The compound microbial agent, *Sphingomyelin-Bacillus basilata* bacterial solution, and *Coccidia fibrosus* bacterial solution from Example 1 were used as experimental samples. Corn stalks were crushed to a length of 4-8 cm for later use.

[0040] 1. Experimental Methods

[0041] Using a saline-alkali soil area with no vegetation in Jinzhong City, Shanxi Province as the experimental site, four non-adjacent plots of 10m × 10m, with a total area of ​​100m², were randomly selected. 2 A square area was designated as the experimental zone. The pH value, salinity, and organic matter content of the soil in each experimental zone were measured to determine that there was no significant difference in the initial state of the soil among the experimental zones.

[0042] Four experimental plots were designated as the corn stalk group, the *Bacillus spp.* group, the *C. cellulose-degrading* group, and the compound microbial agent group, respectively. 36 kg of crushed corn stalks were evenly applied to each plot. The corn stalk group was sprayed with 9 L of sterile water; the *Bacillus spp.* group was sprayed with 9 L of *Bacillus spp.* bacterial solution; the *C. cellulose-degrading* group was sprayed with 9 L of *C. cellulose-degrading* bacterial solution; and the compound microbial agent group was sprayed with 9 L of compound microbial agent. The soil in each experimental plot was thoroughly tilled. Samples were taken after 30 days and 180 days to test the soil pH, salinity, and organic matter content.

[0043] 2. Experimental Results

[0044] The results are shown in Table 1. After 30 days of treatment, each treatment group showed varying degrees of improvement on saline-alkali soil. Compared with the corn straw group, the *Bacillus sphingolipidae* group and the *C. cytotoxicum* group showed significantly better improvement in pH, salinity, and organic matter content of saline-alkali soil. Compared with the corn straw group, the compound microbial agent group significantly improved the pH, salinity, and organic matter content of saline-alkali soil, and the effect was better than that of the *Bacillus sphingolipidae* group or the *C. cytotoxicum* group, demonstrating that the compound microbial agent in this invention exerted a synergistic improvement effect of the two microorganisms.

[0045] After 180 days of treatment, the improvement effect of each treatment group weakened to varying degrees, and the pH value and salinity index reverted to the initial soil state. Compared with the *Bacillus sphingolipidus* group or the *Coccidioidomyces fibronectin* group, the compound microbial agent group had a significantly more sustained and stable effect on improving saline-alkali soil.

[0046] Table 1. Statistics on soil pH, salinity, and organic matter content in each experimental area.

[0047]

[0048] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A microbial composition for remediating saline-alkali soil, characterized in that, This includes *Sphingobacterium bambusae* and *Deinococcus cellulosilyticus*.

2. The application of the microbial composition as described in claim 1 in the preparation of a compound microbial agent for remediating saline-alkali soil.

3. A method for preparing a compound microbial agent for remediating saline-alkali soil, characterized in that, Includes the following steps: After activating the *Sphingomyelin-Bacillus sphingolipidus* and *Coccidioidomyces fibronectin* as described in claim 1, they are fermented, centrifuged, the precipitate is collected and resuspended to obtain a bacterial solution; the bacterial solution is mixed to obtain the compound bacterial agent.

4. The preparation method according to claim 3, characterized in that, The ratio of viable bacteria of *Sphingomonas sphingolipidae* to viable bacteria of *Coccidioidomyces fibronectin* is 1:

1.

5. The preparation method according to claim 3 or 4, characterized in that, In the compound bacterial agent, the total viable count of *Sphingomyelin-Bacillus sphingolipidae* and *Coccidia fibrolytica* is not less than 1 × 10⁻⁶. 8 CFU / mL.

6. A compound microbial agent obtained by the preparation method according to any one of claims 3-5.

7. The application of the microbial composition of claim 1 or the compound microbial agent of claim 6 in the remediation of saline-alkali soil.

8. A method for remediating saline-alkali soil, characterized in that, Includes the following steps: The corn stalks are crushed and applied evenly to the saline-alkali soil. The compound microbial agent described in claim 6 is then sprayed evenly onto the surface of the crushed corn stalks, followed by tilling.

9. The method as described in claim 8, characterized in that, The volume-to-mass ratio of the compound microbial agent to the corn stalk is 1L:4kg.

10. The method as described in claim 8, characterized in that, The application rate of corn stalks is 3600 kg / hm². 2 .

Images