Composition for saline-alkali soil remediation as well as preparation method and application thereof

Abstract

The invention discloses a composition for saline-alkali soil remediation. The composition comprises the following components in percentage by mass: 0.1%-1% of single rhamnolipid, 0.01%-0.1% of polyglutamic acid and 0.1%-1% of pantoea polysaccharide. Results of the application show that the addition of the single rhamnolipid can promote the formation of large aggregates compared with the addition of double rhamnolipid, which indicates that the single rhamnolipid plays an important role in improving the stability and water binding capacity of soil aggregates, and the saline-alkali soil remediation solvent prepared from the single rhamnolipid, polyglutamic acid and pantoea polysaccharide as raw materials has the advantages that the saline-alkali soil remediation effect is good; the method has the advantages of high activity, high efficiency and no secondary pollution risk on the saline-alkali soil remediation function.

Description

Technical Field

[0001] The invention relates to the technical field of production of saline-alkali land restoration preparations, and in particular to a composition for saline-alkali land restoration, a preparation method and application thereof. Background Art

[0002] Soil salinization is a major problem facing soil degradation and one of the main threats to soil degradation worldwide. Many areas use salt water for irrigation, and the evaporation of water has greatly aggravated the problem of salinization in arid and semi-arid soils. Traditional methods of improving saline-alkali soils mainly include leaching and deep plowing. Leaching is the simplest method, which uses fresh water to flush saline-alkali soil to reduce soil salinity, but it consumes a lot of fresh water, which is too costly for saline-alkali areas that are already short of water. Deep plowing mixes surface soil and deep profile soil, which can effectively reduce salinity, but it is very destructive to the soil and can easily aggravate salinization over a long period of time. In addition, adding chemical amendments is also a common method, but it is easy to cause the loss of water, soil and nutrients, and there is a possibility of secondary pollution.

[0003] Many research results show that rhamnolipids can alleviate the accumulation of salt in the surface soil caused by soil capillary action, alleviate salt stress in saline-alkali land, and improve plant resistance and reduce salt damage. It has broad application prospects in the fields of regulation and fertilizer efficiency in plant cultivation. For example, CN113892317A discloses the application of rhamnolipids in the treatment of saline-alkali soil and the improvement of cotton yield in saline-alkali land. The present invention first finds that rhamnolipids can be used to treat saline-alkali land. Secondly, the present invention drips the rhamnolipid aqueous solution to the cotton root zone of saline-alkali land in a timely and quantitative manner through a drip irrigation system. Compared with the existing saline-alkali soil improvement method, this method can directly transport the soil conditioner to the crop root zone environment, achieving the purpose of precise improvement and cost saving. CN116199540A discloses an amino acid-rhamnolipid mixed solution for improving the salt tolerance of crops and a preparation method thereof, wherein amino acid fertilizer is used as a mother solution, and an auxiliary agent rhamnolipid is added, and the two are matched to form a mixed solution; the present invention also provides an application method of the amino acid-rhamnolipid mixed solution, through which the use of the mixed solution can not only promote the development of plant nutritional functions, but also regulate the resistance of crops, improve the salt tolerance of crops in saline soil, thereby promoting the growth of crops in saline soil, improving yield and quality, and realizing the improvement of saline soil. However, at present, it is not clear whether monorhamnolipid or dirhamnolipid plays the main role. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a composition for saline-alkali land restoration and its preparation method and application in view of the deficiencies in the prior art. The compositions are all based on biosynthetic products and have the advantages of strong activity, high efficiency and no risk of secondary pollution.

[0005] In order to solve the above technical problems, the present invention discloses a composition for saline-alkali land restoration, the composition comprising the following components in mass percentage: 0.1%-1% of monorhamnolipid, 0.01%-0.1% of polyglutamic acid and 0.1%-1% of pantoea polysaccharide, wherein the monorhamnolipid is prepared by the following method: a single colony of Pseudomonas aeruginosa with a preservation number of CGMCC NO.29584 is cultured in LB medium to obtain seed liquid, and then fermented in a fermentation medium with vegetable oil as a carbon source to obtain rhamnolipid fermentation liquid, and then separated and purified from the rhamnolipid fermentation liquid to obtain monorhamnolipid.

[0006] Specifically, the steps of separating and purifying monorhamnolipid from rhamnolipid fermentation broth are as follows: sterilizing the rhamnolipid fermentation broth, collecting the supernatant by centrifugation, sterilizing, and then adjusting the pH of the supernatant to 1.5-3.0, standing for 8-16 h, collecting the precipitate by centrifugation, suspending the precipitate with 0.04-0.06 mol / L NaHCO3 solution for several times, collecting the supernatant by centrifugation again, adjusting the pH value of the supernatant to 1.5-3.0, standing for precipitation, and finally extracting the obtained precipitate with ethyl acetate, collecting the ethyl acetate organic phase, mixing the organic phase with a phosphate buffer solution with a pH of 7.5-8.5, and enriching the rhamnolipid in the organic phase in the aqueous phase. Collecting the lower aqueous phase to obtain a rhamnolipid solution, it should be noted that the monorhamnolipid described in the present application is not a pure monorhamnolipid, but a monorhamnolipid with a relatively high mass of the total rhamnolipid. Specifically, the mass ratio of the monorhamnolipid to the total rhamnolipid is greater than 75%.

[0007] The fermentation medium is composed of: a total carbon source concentration of 70-90 g / L, a total nitrogen source concentration of 4-10 g / L, a phosphate concentration of 4-8 g / L, a total magnesium ion concentration of 0.2-1.0 g / L, and a total trace element concentration of 2-6 ml / L; further, the nitrogen source is sodium nitrate; the phosphate is a combination of KH2PO4 and Na2HPO4; the magnesium ion is magnesium sulfate; the trace element is a combination of FeCl3, ZnSO4, CuSO4, MnSO4, and H3BO3; further, the fermentation medium is composed of: 80 g / L rapeseed oil; 8 g / L sodium nitrate; 3.6 g / L Na2HPO4; 2.4 g / L KH2PO4; 0.5 g / L MgSO4; and 4 ml / L trace element mother liquor, wherein the trace element mother liquor is prepared by mixing 0.16 g FeCl3, 1.5 g ZnSO4, and 0.15 g CuSO4. g, 1.5 g MnSO4, and 1.5 g H3BO3 were dissolved in 1 L deionized water, the pH value was adjusted to 2.0, and the mixture was obtained without sterilization and refrigerated at 4 °C for use.

[0008] Specifically, the polyglutamic acid is prepared by the following preparation method: the polyglutamic acid fermentation liquid is first adjusted to a pH of 2-5 with sulfuric acid, 0.1-3wt% diatomaceous earth is added and stirred evenly, sterilized by plate and frame filtration, heated at 90-120° C. for 0.5-3h, and then the pH is adjusted to 6.0-8.0 with 30-40wt% NaOH to obtain a polyglutamic acid solution; the polyglutamic acid fermentation liquid is obtained by fermentation of Bacillus subtilis with a preservation number of CGMCC NO.0833.

[0009] The polyglutamic acid fermentation broth is prepared by the following steps: selecting a single colony of Bacillus subtilis with a preservation number of CGMCC NO.0833 in a fresh seed culture medium, culturing for 14-16 hours under the shaking conditions of 32° C. and 200 r / min, transferring the seed liquid to the fermentation culture medium at an inoculum volume ratio of 3-5%, the fermentation temperature is 28-37° C., the pH is 6.0-7.5, the ventilation volume is 0.5-1.5 vvm, and the stirring speed is 300-1000 r / min; when the total sugar remaining in the fermentation broth is 10 g / L, adding the feed fermentation culture medium to the fermentation broth, controlling the sugar concentration in the fermentation broth to be maintained at about 10-15 g / L, performing fed-batch fermentation, and fermenting for 70-72 hours to obtain the polyglutamic acid fermentation broth, wherein the fermentation culture medium is composed of 40-45 g glucose and 25 g / L sodium bicarbonate. g / L sodium glutamate 35~45g / L, peptone 2~4g / L, (NH4)2SO44~6 g / L, K2HPO4•3H2O 8~12g / L, KH2PO40.1~1g / L, MgSO4•7H2O 0.1~0.5g / L, pH 6.5~7.5; the feed medium is 40-45 g / L glucose.

[0010] The pantoea polysaccharide is prepared by the following method: subjecting the fermentation liquid containing extracellular polysaccharides obtained by fermenting Pantoea camelthorn with a preservation number of CGMCC NO. 15526 to high temperature treatment to obtain a material after the first viscosity reduction; transporting the material after the first viscosity reduction to a high-pressure homogenizer through a pipeline, and subjecting the material to a wall breaking treatment by the high-pressure homogenizer to obtain a material after the second viscosity reduction; filtering the material after the second viscosity reduction through a plate and frame filter press to remove bacterial fragments to obtain a clarified polysaccharide solution; and concentrating the filtrate to obtain a pantoea polysaccharide solution; Among them, the high temperature treatment is to treat the fermentation liquid in an oil bath at 100-120°C for 5-30 minutes; the high-pressure homogenizer wall breaking treatment is a pressure of 300-1000 bar and a flow rate of 600L / h; the concentration is to remove more than 2 / 3vt% of the original volume of water in the filtrate.

[0011] The preparation method of the fermentation liquid containing extracellular polysaccharides is as follows: a single colony of Pantoea serrata with a preservation number of CGMCC NO. 15526 is selected and cultured in a fresh LB liquid culture medium to obtain a seed liquid, and the seed liquid is transferred to a fermentation medium at an inoculation amount of 3-5% by volume, and then the fermentation process is controlled at a temperature of 25-32° C., a ventilation intensity of 0.1-0.8 vvm, a pH of 6.5-7.5, and a stirring rate of 600-800 rpm for 16-48 hours to obtain a fermentation liquid containing extracellular polysaccharides; wherein the fermentation medium comprises a carbon source, a nitrogen source, an inorganic salt and an oxygen carrier, wherein the carbon source is any one of sucrose and molasses or a combination of two of them, and the total concentration of the carbon source is 35-45 g / L; the nitrogen source is any one of corn steep liquor and peptone or a combination of two of them, and the total concentration of the nitrogen source is 3-5 g / L; the inorganic salt is K2HPO4, and the total concentration of the inorganic salt is 1 .5~2g / L; the oxygen carrier contains 2.33g / L n-hexane, 5.36g / L n-heptane, and 9.06g / L n-hexadecane.

[0012] The present invention further proposes a method for preparing the above-mentioned composition, wherein the mother liquors of the above-prepared monorhamnolipid solution, polyglutamic acid solution and pantothenate polysaccharide solution are weighed according to the components in the formula, mixed with water, and stirred sufficiently to obtain the composition.

[0013] The invention also proposes the application of the composition in saline-alkali land restoration.

[0014] When used, the composition is added to the soil in an amount of 1-5% by mass, and water is replenished regularly to keep the soil moisture content at 3wt%-7wt%.

[0015] The composition reduces the conductivity and pH value of saline-alkali soil, promotes the formation of large soil aggregates, and improves the stability and water holding capacity of soil aggregates.

[0016] Beneficial effects: Rhamnolipid is a biosurfactant produced by the metabolism of Pseudomonas and Burkholderia, and belongs to the glycolipid anionic surfactant. It can combine with water molecules, reduce soil water evaporation, enhance wettability and water transmission, and ionize H+ under alkaline conditions to neutralize OH- in alkaline soil, showing great potential in sudden saline-alkali repair. The prepared rhamnolipid of the present application has a high content of monorhamnolipid. Using it as a raw material can effectively improve the application of rhamnolipid in plant stress resistance.

[0017] Polyglutamic acid (γ-PGA) is a high-molecular biomaterial obtained by microbial fermentation. It is a special anionic natural polymer that can chelate metal cations through amine bonds and carboxyl groups. It has excellent water absorption capacity, which helps to increase soil water holding capacity. It is water-soluble, non-toxic and biodegradable, making it an important new raw material in agricultural production. Pantoea polysaccharide is a heteropolysaccharide obtained by fungal fermentation. It is a new type of biostimulant with good hygroscopicity and moisture retention. It can activate soil nutrients and improve soil ecological balance. The saline-alkali land remediation solvent prepared from rhamnolipids, polyglutamic acid and pantoea polysaccharide has the advantages of strong activity, high efficiency and no secondary pollution risk for saline-alkali soil remediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a rhamnolipid TLC plate image; Figure 2 This is a schematic diagram of soil aggregate structure analysis; Figure 3 Schematic diagram of a rice seedling plant.

[0019] The strains involved in this application are: (1) Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) The strain name is ST3, the deposit number is CGMCCNO.29584, the deposit date is January 11, 2024, and the deposit location is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101.

[0020] (2) Pantotrichum camelthorn P.alhagi XK-11, deposit number is CGMCC NO. 15526. Detailed information of the strain is disclosed in CN111808777A.

[0021] (3) Bacillus subtilis NX-2, with a deposit number of CGMCC NO.0833, and its detailed information is disclosed in CN1164737C. DETAILED DESCRIPTION

[0022] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0023] Example 1 Preparation of rhamnolipid, pantothenate polysaccharide and polyglutamic acid.

[0024] (A) Preparation of dirhamnolipid solution.

[0025] The specific steps are as follows: (1) Preparation method of rhamnolipid fermentation broth: A single colony of Pseudomonas aeruginosa (P. aeruginosa ST3, with a collection number of CGMCC NO. 29584) was selected and inoculated into a fresh 500 ml Erlenmeyer flask pre-filled with 100 ml of LB liquid medium, and cultured at 37°C and 200 rpm for 16 hours to obtain a seed solution. The seed solution was cultured in a 5 L fermentation tank pre-filled with 3.0 L of sterilized fermentation medium at an inoculation rate of 4% (v / v). Subsequently, the fermentation process was controlled at a temperature of 37°C, a pH of 7.0, a ventilation rate of 1.0 vvm, a stirring speed of 300 rpm, and fermented for 72-120 hours to obtain a rhamnolipid fermentation broth.

[0026] The Pseudomonas aeruginosa was screened by the inventor from oily swill and oilfield soil in the cafeteria of Nanjing University of Technology in July 2023. The 16S rDNA of Pseudomonas aeruginosa ST3 is shown in the sequence table, and the full length of the sequence is 1327bp. By comparing the blastN sequence comparison tool in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), the strain of the present invention was identified as Pseudomonas aeruginosa and named ST3.

[0027] Seed culture medium (LB liquid medium): peptone 10 g / L, NaCl 10 g / L, yeast extract 5 g / L.

[0028] Fermentation medium: 40g / L glycerol; 8g / L sodium nitrate; 3.6g / L Na2HPO4; 2.4g / L KH2PO4; 0.5g / LMgSO4; 4ml / L trace element mother solution.

[0029] (2) Preparation of rhamnolipid solution: The preparation steps refer to the steps in "Research on the production of rhamnolipids by Pseudomonas aeruginosa 1.10452 and its efficient purification and antibacterial activity" (Yang Huan. Research on the production of rhamnolipids by Pseudomonas aeruginosa 1.10452 and its efficient purification and antibacterial activity [D]. South China University of Technology, 2021. DOI: 10.27151 / d.cnki.ghnlu.2021.001140.), the rhamnolipid fermentation broth obtained in step (1) was sterilized, centrifuged at 8000 rpm for 10 min, the bacteria were removed, and then the supernatant was collected, and the pH value of the sterilized fermentation broth was adjusted to 2.0 with 6 mol / L HCl, and the precipitate was collected by centrifugation at 8000 rpm for 10 min. The precipitate was suspended with 0.05 mol / L NaHCO3 solution for 3 times and centrifuged (8000 rpm, 10 min) and collect the supernatant. The pH value of the supernatant was adjusted to 2.0 and allowed to settle. Finally, the precipitate was extracted with an equal volume of ethyl acetate, and the ethyl acetate organic phase was collected. The organic phase was mixed with a pH 8 phosphate buffer solution (pH 7.8-8.0) in an equal volume ratio. The rhamnolipid in the organic phase was enriched in the aqueous phase. The lower aqueous phase was collected to obtain a solution rich in rhamnolipid, which was recorded as rhamnolipid-GS.

[0030] 2. Preparation of monorhamnolipid The monorhamnolipid fermentation broth is obtained by fermenting Pseudomonas aeruginosa. The preparation method of the monorhamnolipid fermentation broth is the same as the preparation method of the disrhamnolipid fermentation broth in step (1), except that the carbon source in the fermentation medium is 80 g / L rapeseed oil, and the remaining components are the same as those in the disrhamnolipid fermentation medium, thereby preparing a solution rich in monorhamnolipid, which is recorded as rhamnolipid-CD.

[0031] The prepared rhamnolipid-GS and rhamnolipid-CD solutions were subjected to thin layer chromatography to detect the proportion of monosaccharide and disaccharide lipids. The results are shown in Table 1. Figure 1 As shown, the proportion of disrhamnolipids in rhamnolipid-GS is 95.4%, and the proportion of monorhamnolipids is 5.5%; the proportion of disrhamnolipids in rhamnolipid-CD is 22.4%, and the proportion of monorhamnolipids is 77.6%.

[0032] Wherein, the detection method of thin layer chromatography is as follows: 1) Prepare the sample solution. Prepare the rhamnolipid-GS solution, rhamnolipid-CD solution, and rhamnolipid standard solution obtained above into a 20 g / L rhamnolipid solution.

[0033] 2) Prepare the developing agent. The developing agent must be prepared immediately before use. Prepare it in the ratio of chloroform: methanol: acetic acid (72:8:2). After adding the solution into the developing cylinder, immediately cover the lid to allow the gas to fill the entire container.

[0034] 3) Prepare the color developer. The color developer must be prepared before use. The color developer is 0.1% anthrone sulfuric acid solution. Weigh 0.05 g of anthrone and add it to 50 ml of concentrated sulfuric acid. Shake gently to mix.

[0035] 4) Spot the sample using a micro capillary and dry it with a hair dryer. Spot about 3μl of sample for chromatography. Immerse the spotted TLC plate in the developing agent at an angle. After the sample is fully developed, dry it naturally in a fume hood.

[0036] 5) Color development: spray the prepared color developer evenly on the surface of the TLC plate, and then place the TLC plate in a constant temperature oven at 80°C for 15 minutes.

[0037] Table 1 Analysis of rhamnolipid spots on TLC plate

[0038] (III) Preparation of Pantoea polysaccharide solution.

[0039] Pantoea polysaccharide fermentation broth Pantoea polysaccharide fermentation broth is obtained by fermenting Pantoea alhagi XK-11 (preservation number: CGMCC NO. 15526).

[0040] Specifically, the preparation method of the pantoea polysaccharide fermentation liquid is as follows: a single colony of P. alhagi XK-11 is selected and placed in a fresh LB liquid culture medium, and cultured overnight at 37°C and 200rpm to obtain a seed liquid. The seed liquid is transferred to the fermentation medium at an inoculation rate of 4% (v / v), and then the fermentation process is controlled at a temperature of 30°C, a pH of 7.0, a ventilation volume of 0.8vvm, a stirring speed of 800rpm, and fermented for 24 hours to obtain the pantoea exopolysaccharide fermentation liquid.

[0041] Fermentation medium: 40 g / L sucrose; 5 g / L peptone; 2 g / L K2HPO4; 2.33 g / L n-hexane, 5.36 g / L n-heptane, 9.06 g / L n-hexadecane.

[0042] The pantoea polysaccharide fermentation broth obtained by fermentation was subjected to an in-situ high-temperature (110°C) oil bath viscosity reduction treatment for 15 minutes. Subsequently, it was once again subjected to a high-pressure homogenizer wall-breaking treatment to reduce viscosity, wherein the pressure was 600 bar and the flow rate was 600L / h, and a plate and frame filter press was used to remove bacterial fragments and other precipitates to obtain a clear polysaccharide solution. The polysaccharide solution was concentrated to 2 / 3 of the original volume by a rotary evaporator to obtain the pantoea polysaccharide solution, with a concentration of 28.9g / L.

[0043] (IV) Preparation of polyglutamic acid solution.

[0044] Preparation of polyglutamic acid fermentation broth: obtained by fermentation of Bacillus subtilis NX-2. The preparation method of the polyglutamic acid fermentation broth is as follows: a single colony of Bacillus subtilis is selected in a fresh seed culture medium, cultured at 32°C, 200r / min under shaking conditions for 16h, and the seed liquid is transferred to the fermentation medium at a seed amount of 3% (v / v). The temperature of the fermentation process is controlled at 32°C, 600r / min, and the ventilation volume is 4.5L / min. When the total residual sugar in the fermentation broth is 10g / L, the feed fermentation medium is added to the fermentation broth, and the sugar concentration in the fermentation broth is controlled to be maintained at about 10-15g / L, and fed-batch fermentation is performed. Ferment for 72h to obtain polyglutamic acid fermentation broth.

[0045] Seed culture medium: Seed culture medium: glucose 5 g / L, beef extract 5 g / L, peptone 5 g / L, MgSO4·7H2O0.5 g / L, pH adjusted to 6.8 with NaOH solution.

[0046] Fermentation medium: total glucose concentration 80 g / L (including 40 g / L initial glucose and the remaining 40 g / L glucose as fed-batch fermentation medium added to the fermentation liquid), sodium glutamate 40 g / L, peptone 3 g / L, (NH4)2SO4 5 g / L, K2HPO4·3H2O 10 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.25 g / L, pH 7.0.

[0047] Preparation method of polyglutamic acid solution: adjust the pH of polyglutamic acid fermentation liquid to 3 with sulfuric acid, add 1.5 wt% diatomaceous earth and stir evenly, sterilize by plate and frame filtration, heat at 100°C for 2h, and then adjust the pH to 7.0 with 35wt% NaOH to obtain the polyglutamic acid solution with a concentration of 39.2g / L.

[0048] The dirhamnolipid and monorhamnolipid solutions prepared above are diluted to 20 g / L with aqueous solution. The stock solutions of Pantoea polysaccharide solution and polyglutamic acid solution can be used as they are.

[0049] Example 2 Composition for saline-alkali land restoration.

[0050] A composition for saline-alkali land restoration comprises rhamnolipid-CD, polyglutamic acid, pantothenate polysaccharide and water, wherein the content of rhamnolipid-CD is 0.76wt%, the content of polyglutamic acid is 0.057wt%, and the content of pantothenate polysaccharide is 0.21wt%.

[0051] Preparation method: Measure the corresponding volume of solution according to the configuration, mix, add the remaining amount of water and stir thoroughly.

[0052] Example 3 A composition for saline-alkali land restoration, which is different from Example 2 in that the rhamnolipid-CD solution is rhamnolipid-GS.

[0053] Comparative Example 1 is the same as Example 2 or 3, except that the raw material does not contain rhamnolipid solution.

[0054] Comparative Example 2 is the same as Example 2, except that the raw material does not contain polyglutamic acid solution.

[0055] Comparative Example 3 is the same as Example 3, except that the raw material does not contain polyglutamic acid solution.

[0056] Comparative Example 4 is the same as Example 2, except that the raw material does not contain Pantoea polysaccharide solution.

[0057] Comparative Example 5 is the same as Example 3, except that the raw material does not contain Pantoea polysaccharide solution.

[0058] Effects of different compositions on improving soil properties in saline-alkali land.

[0059] The saline-alkali land restoration solvents prepared in Examples 2-3 and Comparative Examples 1-5 and the composition prepared in Example 1 were stored at room temperature for 1 month and then subjected to experiments, and tap water was used as a blank control group for saline-alkali land restoration. A total of 8 groups of experiments were set up, and AH were respectively Examples 2-3, Comparative Examples 1-5, and tap water.

[0060] 500 g of saline-alkali soil was added with (1) 3 wt% sterilized water and (2) 3 wt% of the above-mentioned saline-alkali land remediation solvent. The soil moisture content was maintained at 5% by regular water replenishment. The soil was left stationary at room temperature for 70 days. On the 0th, 14th, 28th, 49th and 70th days, soil samples at a depth of 6 cm were collected for further analysis.

[0061] (1) Determination of soil salinity The improvement effect of soil salinity is expressed by electrical conductivity (EC) and pH. The specific method is: the soil sample is naturally air-dried and then crushed, and passed through a 18-mesh sieve. 10g of the sieved soil is weighed, 50ml of deionized water is added, stirred and left to stand for 30 minutes, and the pH and electrical conductivity (EC) are measured.

[0062] (2) Soil aggregate structure The soil samples were broken apart along natural cracks and air-dried. The air-dried soil was placed on sieves with apertures of 5 mm, 1 mm and 0.25 mm, and shaken up and down for 2 minutes to remove soil aggregates larger than 5 mm, 1-5 mm, 0.25-1 mm and smaller than 0.25 mm. Soil samples at different levels were weighed.

[0063] The electrical conductivity (EC) and pH value of the saline-alkali land after restoration by the restoration solvent were detected and counted. The results on the 70th day are shown in Table 2, where A-H are the result values ​​of Examples 2-3, Comparative Examples 1-5, and tap water, respectively. The soil EC value and pH value of Group A after restoration were significantly lower than those of the other groups, indicating that the addition of rhamnolipid can effectively reduce the EC value and pH value of saline-alkali land.

[0064] Table 2 Soil EC value and pH value on day 70

[0065] The aggregate structure of the repaired soil was statistically analyzed, and the results are as follows: Figure 2 As shown, the addition of monorhamnolipids-rich soil can promote the formation of large aggregates, indicating that monorhamnolipids play an important role in improving the stability of soil aggregates and water holding capacity.

[0066] Effect verification example 2 Rice was used as the experimental crop, and a total of 8 groups of experiments were set up, with AH being Examples 2-3, Comparative Examples 1-5, and tap water. Before planting, the saline-alkali soil was first repaired with the configured repair solvent, and 5 kg / mu of the repair solvent was sprayed on the saline-alkali land to be repaired every 7 days, for a total of 10 times to repair the saline-alkali land. Crops (rice) were planted in the repaired soil, and watering, pest control and fertilization were carried out according to conventional methods during the planting period.

[0067] The growth of rice was observed. Among them, the rice plants in group A had the best growth situation and the rice seedlings in group A had the longest root length. Figure 3 As shown; and the biomass of group A was the highest, 23% higher than that of group H. The results are shown in Table 3, indicating that the addition of monorhamnose can effectively promote the growth of rice in saline-alkali land.

[0068] Table 3 Experimental crop yield (kg / mu)

[0069] In summary, the mixed saline-alkali land remediation solvent rich in monorhamnolipid, polyglutamic acid and pantothecoid polysaccharide has a highly efficient and high-quality remediation function for saline-alkali land remediation.

[0070] The present invention provides a composition and method for saline-alkali land restoration. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A composition for saline-alkali land restoration, characterized in that: The composition comprises the following components in percentage by mass: 0.1%-1% monorhamnolipid, 0.01%-0.1% polyglutamic acid and 0.1%-1% pantoea polysaccharide, wherein the monorhamnolipid is prepared by the following method: a single colony of Pseudomonas aeruginosa with a preservation number of CGMCC NO.29584 is cultured in LB medium to obtain seed liquid, and then fermented in a fermentation medium with vegetable oil as a carbon source to obtain rhamnolipid fermentation liquid, and then separated and purified from the rhamnolipid fermentation liquid to obtain monorhamnolipid.

2. The composition according to claim 1, characterized in that The steps of separating and purifying monorhamnolipid from rhamnolipid fermentation broth are as follows: sterilizing the rhamnolipid fermentation broth, collecting the supernatant by centrifugation, sterilizing the supernatant, then adjusting the pH of the supernatant to 1.5-3.0, standing for 8-16 hours, collecting the precipitate by centrifugation, suspending the precipitate with a 0.04-0.06 mol / L NaHCO3 solution for several times, collecting the supernatant by centrifugation again, adjusting the pH of the supernatant to 1.5-3.0, standing for collecting the precipitate, and finally extracting the precipitate with ethyl acetate, collecting the ethyl acetate organic phase, mixing the organic phase with a phosphate buffer solution with a pH of 7.5-8.5, enriching the rhamnolipid in the organic phase in the aqueous phase, and collecting the lower aqueous phase is an aqueous solution containing monorhamnolipid.

3. The composition according to claim 2, characterized in that The fermentation medium is composed of: a total carbon source concentration of 70-90 g / L, a total nitrogen source concentration of 4-10 g / L, a phosphate concentration of 4-8 g / L, a total magnesium ion concentration of 0.2-1.0 g / L, and a total trace element concentration of 2-6 ml / L, wherein the carbon source is any one of rapeseed oil, corn oil, peanut oil, and soybean oil, the nitrogen source is sodium nitrate, the phosphate is a combination of KH2PO4 and Na2HPO4, the magnesium ion is magnesium sulfate, and the trace element is a combination of FeCl3, ZnSO4, CuSO4, MnSO4, and H3BO3.

4. The composition according to claim 1, characterized in that The polyglutamic acid is prepared by the following preparation method: the polyglutamic acid fermentation liquid is first adjusted to a pH of 2-5 with sulfuric acid, 0.1-3wt% diatomaceous earth is added and stirred evenly, the polyglutamic acid solution is obtained by plate-frame filtration, heating at 90-120° C. for 0.5-3h, and then adjusting the pH to 6.0-8.0 with 30-40wt% NaOH; the polyglutamic acid fermentation liquid is obtained by fermentation of Bacillus subtilis with a preservation number of CGMCC NO.0833, wherein the polyglutamic acid fermentation liquid is prepared by the following steps: a single colony of Bacillus subtilis with a preservation number of CGMCC NO.0833 is selected and placed in a fresh seed culture medium, and cultured at 32° C. and 200r / min under shaking conditions for 14-16h, and the seed liquid is transferred to the fermentation culture medium at an inoculum amount of 3-5% by volume, and the fermentation temperature is 28-37° C., the pH is 6.0-7.5, and the ventilation volume is 0.5 ~ 1.5vvm, the stirring speed is 300 ~ 1000r / min; when the residual total sugar in the fermentation broth is 10g / L, the fed-batch fermentation medium is added to the fermentation broth, the sugar concentration in the fermentation broth is controlled to be maintained at 10-15g / L, and the fed-batch fermentation is carried out for 70-72h to obtain a polyglutamic acid fermentation broth, wherein the fermentation medium is composed of: 40-45 g / L glucose, 35~45g / L sodium glutamate, 2~4g / L peptone, 4~6 g / L (NH4)2SO4, 8~12g / L K2HPO4•3H2O, 0.1~1g / L KH2PO4, 0.1~0.5g / L MgSO4•7H2O, pH 6.5~7.5; the feed medium is 40-45 g / L glucose.

5. The composition according to claim 1, characterized in that The pantoea polysaccharide is prepared by the following method: subjecting the fermentation liquid containing extracellular polysaccharides obtained by fermenting Pantoea camelthorn with a preservation number of CGMCC NO. 15526 to high temperature treatment to obtain a material after the first viscosity reduction; transporting the material after the first viscosity reduction to a high-pressure homogenizer through a pipeline, and subjecting the material to a wall breaking treatment by the high-pressure homogenizer to obtain a material after the second viscosity reduction; filtering the material after the second viscosity reduction through a plate and frame filter press to remove bacterial fragments to obtain a clarified polysaccharide solution; and concentrating the filtrate to obtain a pantoea polysaccharide solution; The high temperature treatment is to treat the fermentation liquid in an oil bath at 100-120°C for 5-30 minutes; the high pressure homogenizer wall breaking treatment is a pressure of 300-1000 bar and a flow rate of 600 L / h; the concentration is to remove more than 2 / 3 vt% of the original volume of water in the filtrate. The preparation method of the fermentation liquid containing extracellular polysaccharides is as follows: a single colony of Pantoea acuminata with a preservation number of CGMCC NO. 15526 is selected and cultured in a fresh LB liquid culture medium to obtain a seed liquid, and the seed liquid is transferred to the fermentation medium at an inoculation rate of 3-5% by volume. Subsequently, the fermentation process is temperature controlled at 25-32°C, the ventilation intensity is 0.1-0.8 vvm, the pH is 6.5-7.5, and the stirring rate is 600-800. rpm for 16 to 48 hours to obtain a fermentation broth containing extracellular polysaccharides; wherein the fermentation medium comprises a carbon source, a nitrogen source, an inorganic salt and an oxygen carrier, wherein the carbon source is any one of sucrose and molasses or a combination of two thereof, and the total concentration of the carbon source is 35 to 45 g / L; the nitrogen source is any one of corn steep liquor and peptone or a combination of two thereof, and the total concentration of the nitrogen source is 3 to 5 g / L; the inorganic salt is K2HPO4, and the total concentration of the inorganic salt is 1.5 to 2 g / L; the oxygen carrier comprises 2 to 3 g / L n-hexane, 5 to 6 g / L n-heptane and 8.5 to 9.5 g / L.

6. A method for preparing the composition according to any one of claims 1 to 5, characterized in that: The monorhamnolipid solution, the polyglutamic acid solution and the pantothenate polysaccharide solution are weighed according to the components in the formula, mixed, and then prepared with water and stirred sufficiently to obtain the composition.

7. Use of the composition according to any one of claims 1 to 5 in saline-alkali land restoration.

8. The use according to claim 7, characterized in that: When used, the composition is added to the soil in an amount of 1-5% by mass, and water is replenished regularly to keep the soil moisture content at 3wt%-7wt%.

9. The use according to claim 8, characterized in that: The composition reduces the conductivity and pH value of saline-alkali soil, promotes the formation of large soil aggregates, and improves the stability and water holding capacity of soil aggregates.

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