Method for cultivating halotolerant bacteria and its application in agriculture

Abstract

The application relates to a cultivating method of salt-tolerant bacteria and agricultural application, and the cultivating method comprises the following steps: S1, mixing straw, mushroom cultivation waste, poultry manure, inorganic salt and culture medium matrix to obtain a biological waste-based culture medium; S2, respectively culturing pythium and bacillus by using the biological waste-based culture medium to obtain a compound bacterial strain liquid; S3, respectively mixing diatomite and the compound bacterial strain liquid, adsorbing and drying to obtain an intermediate product; S4, blending chitosan, phytanic acid and an activator, and reacting to obtain hydrophobic chitosan; and S5, blending and stirring the intermediate product and the hydrophobic chitosan to obtain salt-tolerant bacteria. The hydrophobic chitosan is beneficial to stable storage of the salt-tolerant bacteria in a high-humidity environment, reduces adverse effects such as growth inhibition between different salt-tolerant bacteria, and makes different salt-tolerant bacteria play a synergistic role, so that the efficiency of the fertilizer is finally improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural technology, in particular to a method for cultivating salt-tolerant bacteria and its application in agriculture. BACKGROUND

[0002] Saline-alkali soil refers to a type of land where excessive accumulation of salt (such as sodium chloride, sodium sulfate, etc.) or alkali (such as sodium carbonate, sodium bicarbonate, etc.) in the soil leads to soil deterioration and affects plant growth. The core characteristic of saline-alkali soil is the excessive salt content in the surface or subsurface layer of the soil (usually more than 0.3%), or a significant increase in pH value (alkali soil pH is usually greater than 9), which leads to serious changes in soil properties and is not suitable for planting crops. The formation of saline-alkali soil is caused by both natural and human factors. Natural factors include drought, strong evaporation, seawater intrusion, land closure, and salt-containing parent material. Human factors include unreasonable irrigation, improper fertilization and tillage, industrialization and urbanization, etc.

[0003] The harm of saline-alkali soil mainly manifests in its negative impact on soil, plants, ecosystems, and human production and life, including: 1. Soil function degradation: soil physical structure is damaged, chemical properties are deteriorated, and microbial activity is inhibited; 2. Agricultural production is damaged: crop production is limited and yields are reduced, and planting costs are increased; 3. Ecological system is damaged: vegetation degradation and biodiversity decline, hydrological and climatic effects; 4. Economic and social impact: threat to food security, high cost of ecological governance, and deterioration of living environment; 5. Long-term environmental risk: secondary salinization spreads and ecological chain is broken.

[0004] Improving and utilizing saline-alkali land can improve low-yield land and increase the arable land area, which is of great significance to maintaining ecological safety and food supply safety. Salt-tolerant bacterial fertilizer is one of the core technologies for biological improvement in saline-alkali land agricultural production. Its significance lies not only in short-term improvement of crop resistance and yield, but also in long-term repair of soil microecology through the action of microorganisms, laying a foundation for the sustainable use of saline-alkali land. This technology is both scientific and economic, and is an important breakthrough in alleviating the global crisis of saline-alkali land and ensuring food security.

[0005] However, there are still some problems in the use of salt-tolerant bacterial fertilizer at present, including: 1. During long-term storage, if the environmental humidity is high, the number of live bacteria may decrease and their activity may decrease, leading to a decrease in the effectiveness of the fertilizer; 2. Different salt-tolerant bacteria may have growth inhibition and other interactions, leading to a decrease in the effectiveness of the fertilizer.

[0006] In view of the above, it is necessary to develop a new technical solution to overcome the defects in the prior art. SUMMARY

[0007] The application provides a cultivation method of salt-tolerant bacteria and agricultural application. The culture medium of the salt-tolerant bacteria of the application takes agricultural waste such as straw, mushroom cultivation waste, poultry manure and inorganic salt as raw materials, and is used for cultivating complex bacterial strains of pythium and bacillus, respectively, adsorbing with diatomite, and wrapping with hydrophobic chitosan, so that the salt-tolerant bacteria can be stably stored in a high-humidity environment, and the growth inhibition and other interactions are reduced, which is beneficial to improving the fertilizer efficiency and has a good application prospect.

[0008] The application aims to provide a cultivation method of salt-tolerant bacteria, which comprises the following steps:

[0009] S1, mixing straw, mushroom cultivation waste, poultry manure, inorganic salt and culture medium matrix to obtain a biological waste-based culture medium;

[0010] S2, cultivating pythium and bacillus respectively by using the biological waste-based culture medium to obtain a complex bacterial strain liquid;

[0011] S3, mixing diatomite and the complex bacterial strain liquid, adsorbing and drying to obtain an intermediate product;

[0012] S4, blending chitosan, phytanic acid and an activating agent, and reacting to obtain hydrophobic chitosan;

[0013] S5, blending and stirring the intermediate product and the hydrophobic chitosan to obtain salt-tolerant bacteria.

[0014] Further, it further comprises:

[0015] S6, mixing the salt-tolerant bacteria with nutrients.

[0016] Further, in step S2, the pythium is selected from oligandrum pythium.

[0017] Further, in step S2, the bacillus is selected from one or more of bacillus subtilis, bacillus thuringiensis and paenibacillus polymyxa.

[0018] Further, in step S1, the inorganic salt is selected from one or more of sodium chloride, potassium chloride and magnesium chloride.

[0019] Further, in step S2, the concentration of the complex bacterial strain liquid is 1-9x10 8 cfu / mL.

[0020] Further, in step S3, the mass ratio of the diatomite to the complex bacterial strain liquid is 1:(1-3).

[0021] Further, in step S4, the mass ratio of the chitosan, phytanic acid and activating agent is (6-10):(1-3):(5-7).

[0022] Further, in step S4, the activating agent is selected from one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N-diisopropylcarbodiimide, N-hydroxysuccinimide.

[0023] Further, in step S5, the mass ratio of the intermediate product and the hydrophobic chitosan is 8:(1-3).

[0024] Further, the nutrient substance comprises nitrogen fertilizer, phosphorus fertilizer and potassium fertilizer.

[0025] Further, the nutrient substance comprises urea, diammonium hydrogen phosphate and potassium sulfate.

[0026] Further, the mass ratio of the salt-tolerant bacteria, urea, diammonium hydrogen phosphate and potassium sulfate is (2-4):(3-6):(4-8):(3-6).

[0027] Further, the use amount ratio of the pythium oligandrum, bacillus subtilis, bacillus thuringiensis and paenibacillus polymyxa is (1-2):(1-2):(1-2):(1-2).

[0028] Another object of the present application is to provide the agricultural application of the cultivation method of the salt-tolerant bacteria.

[0029] The present application has the following beneficial effects:

[0030] The cultivation method of the salt-tolerant bacteria provided by the present application uses agricultural waste materials such as straw, mushroom cultivation waste, poultry manure and inorganic salt as raw materials to cultivate pythium oligandrum, bacillus subtilis, bacillus thuringiensis and paenibacillus polymyxa, obtains a compound bacterial strain liquid, adsorbs the compound bacterial strain liquid by diatomite, and then wraps the compound bacterial strain liquid by hydrophobic chitosan to obtain salt-tolerant bacteria. The salt-tolerant bacteria can be mixed with nutrient substances to obtain a fertilizer. The hydrophobic chitosan is obtained by activating the carboxyl group on phytanic acid by an activating agent and then reacting with the amino group of chitosan, so that an alkyl acid containing multiple branches is introduced into the chitosan. Then, the hydrophobic chitosan is blended with an intermediate product to generate hydrogen bonds and other interactions, so that the hydrophobic chitosan is wrapped on the surface of the intermediate product to form a protective layer. The alkyl acid containing multiple branches makes the chitosan have hydrophobic properties, which is conducive to the stable storage of the salt-tolerant bacteria in a high-humidity environment. The protective layer formed by the hydrophobic chitosan on the surface of the intermediate product avoids direct contact between different salt-tolerant bacteria, reduces the adverse effects such as growth inhibition between different salt-tolerant bacteria, and enables different salt-tolerant bacteria to play a synergistic role, thereby improving the effectiveness of the fertilizer.

[0031] The diatomite has rich pore structure and good adsorption effect, and is used as a carrier of the salt-tolerant bacteria to fix the salt-tolerant bacteria, which is conducive to the preservation and growth of the salt-tolerant bacteria.

[0032] The agricultural waste materials used in the application, such as straw, mushroom cultivation waste, poultry manure and the like, contain rich nutritional components such as cellulose, starch, protein, amino acids and the like, and the salt-tolerant bacteria are cultivated by taking these agricultural waste materials as raw materials, which not only realizes the utilization of the waste materials, but also avoids the pollution of the waste materials to the environment, and is conducive to the sustainable development of agriculture. In addition, the added inorganic salt can simulate the salt stress environment, adjust the osmotic pressure, screen the salt-tolerant strains, maintain the cell function and induce the secretion of metabolic products, so as to ensure that the salt-tolerant bacteria maintain their salt-tolerant characteristics and functional activity during the cultivation process, and finally ensure their application effect in the saline-alkali environment. DETAILED DESCRIPTION

[0033] In order to more clearly illustrate the technical solutions of the present application, the following examples are listed. The raw materials, reactions and post-treatment means appearing in the examples are all common raw materials on the market and technical means familiar to those skilled in the art, unless otherwise stated.

[0034] The words "preferred", "preferably", "more preferred" and the like in the present application refer to the embodiments of the present application which can provide certain beneficial effects in certain cases. However, other embodiments can also be preferred in the same or other cases. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not usable, nor is it intended to exclude other embodiments from the scope of the present application.

[0035] It should be understood that, except in any operating examples, or otherwise indicated, expressions of amount or all numbers, such as, for example, in the specification and claims, using the terms "approximately" or "about" with respect to a particular value, should be understood to refer to the value being modified to be within a range of values that one of ordinary skill in the art would consider equivalent to the value being modified in light of the disclosure. Thus, unless otherwise indicated, the numerical parameters set forth in the following description and associated claims are approximations that can vary depending on the desired properties sought to be obtained by the present application.

[0036] The bacillus subtilis (GDMCC NO. 1.131), bacillus thuringiensis (GDMCC NO. 1.126) and paenibacillus polymyxa (GDMCC NO. 1.1630) in the embodiments of the present application are purchased from Guangdong Microbial Culture Collection Center, and pythium oligandrum (GSICC 61615) is purchased from Gansu Provincial Industrial Microbial Culture Collection Center.

[0037] The straw (corn straw), mushroom cultivation waste (mushroom residue) and poultry manure (cow dung) in the embodiments of the present application are purchased from local markets and farms, and are sterilized before use.

[0038] The culture medium matrix in the embodiment of the application is composed of the following components: beef extract 3.0g, peptone 5.0g, glucose 10.0g, sodium chloride 1.0g, potassium dihydrogen phosphate 0.5g, distilled water 1.0L, and the pH is adjusted to 7.0.

[0039] The chitosan in the embodiment of the application has an average molecular weight of 200,000 and a degree of deacetylation greater than 80%.

[0040] The activator in the embodiment of the application is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0041] The inorganic salt in the embodiment of the application is NaCl and KCl in a mass ratio of 1:1.

[0042] The "parts" in the embodiment of the application all refer to mass parts.

[0043] Embodiment 1

[0044] A method for cultivating a salt-tolerant bacterium, comprising the following steps:

[0045] S1, 3 parts of straw, 2 parts of mushroom cultivation waste, 0.2 parts of poultry manure, and 2 parts of inorganic salt are mixed, dried, crushed through a 100-mesh sieve, and mixed with 10 parts of culture medium matrix to obtain a biological waste-based culture medium;

[0046] S2, the activated Bacillus subtilis, Bacillus thuringiensis, and Paenibacillus polymyxa are respectively added to the biological waste-based culture medium for expansion culture, and the culture is carried out at 30 DEG C for 48h, and the concentration of the bacterial liquid is adjusted to 1x10 8 cfu / mL to obtain a Bacillus subtilis liquid, a Bacillus thuringiensis liquid, and a Paenibacillus polymyxa liquid;

[0047] The activated Pythium oligandrum is added to the biological waste-based culture medium for expansion culture, and the culture is carried out at 26 DEG C for 72h, and the concentration of the bacterial liquid is adjusted to 1x10 8 cfu / mL to obtain a Pythium oligandrum liquid;

[0048] S3, diatomite and the Bacillus subtilis liquid are mixed in a mass ratio of 1:1, uniformly stirred, and then left to stand for 2h, and dried to obtain an intermediate product 1;

[0049] Diatomite and the Bacillus thuringiensis liquid are mixed in a mass ratio of 1:1, uniformly stirred, and then left to stand for 2h, and dried to obtain an intermediate product 2;

[0050] Diatomite and the Paenibacillus polymyxa liquid are mixed in a mass ratio of 1:1, uniformly stirred, and then left to stand for 2h, and dried to obtain an intermediate product 3;

[0051] Mixing diatomite and pythium oligandrum liquid with a mass ratio of 1:1, stirring uniformly, standing for 2h, and drying to obtain intermediate product 4;

[0052] S4, blending chitosan, phytanic acid, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride, N-hydroxysuccinimide, dimethyl sulfoxide in a mass ratio of 8:2:3:3:1000, stirring at 15℃ for 0.5h, heating to 65℃, stirring for 12h, cooling to 20℃, adding isopropyl alcohol (the mass ratio of dimethyl sulfoxide and isopropyl alcohol is 1:1), stirring for 0.5h, filtering, washing, and drying to obtain hydrophobic chitosan;

[0053] S5, blending hydrophobic chitosan and dimethyl sulfoxide in a mass ratio of 1:48, heating to 40℃, stirring for 1h, cooling to room temperature, adding intermediate product 1 (the mass ratio of intermediate product 1 and hydrophobic chitosan is 8:3) and stirring for 1h, standing for 6h, and washing and drying to obtain coated strain 1;

[0054] Blending hydrophobic chitosan and dimethyl sulfoxide in a mass ratio of 1:48, heating to 40℃, stirring for 1h, cooling to room temperature, adding intermediate product 2 (the mass ratio of intermediate product 2 and hydrophobic chitosan is 8:3) and stirring for 1h, standing for 6h, and washing and drying to obtain coated strain 2;

[0055] Blending hydrophobic chitosan and dimethyl sulfoxide in a mass ratio of 1:48, heating to 40℃, stirring for 1h, cooling to room temperature, adding intermediate product 3 (the mass ratio of intermediate product 3 and hydrophobic chitosan is 8:3) and stirring for 1h, standing for 6h, and washing and drying to obtain coated strain 3;

[0056] Blending hydrophobic chitosan and dimethyl sulfoxide in a mass ratio of 1:48, heating to 40℃, stirring for 1h, cooling to room temperature, adding intermediate product 4 (the mass ratio of intermediate product 4 and hydrophobic chitosan is 8:3) and stirring for 1h, standing for 6h, and washing and drying to obtain coated strain 4;

[0057] Blending coated strain 1, coated strain 2, coated strain 3, and coated strain 4 in a mass ratio of 1:1:1:1 to obtain a salt-tolerant bacteria.

[0058] Example 2

[0059] A method for cultivating a salt-tolerant bacteria, the method comprising the following steps:

[0060] S1, mixing 3 parts of straw, 2 parts of mushroom cultivation waste, 0.2 parts of poultry manure, and 2 parts of inorganic salt, drying, crushing through a 100-mesh sieve, and mixing with 10 parts of a culture medium base to obtain a biological waste-based culture medium;

[0061] S2, the activated Bacillus subtilis, Bacillus thuringiensis, Paenibacillus polymyxa are added into the biological waste-based culture medium respectively for expansion culture, and cultured at 30℃ for 48h, and the concentration of the bacterial liquid is adjusted to 1×10 8 cfu / mL, to obtain Bacillus subtilis liquid, Bacillus thuringiensis liquid and Paenibacillus polymyxa liquid;

[0062] The activated Pythium oligandrum is added into the biological waste-based culture medium for expansion culture, and cultured at 26℃ for 72h, and the concentration of the bacterial liquid is adjusted to 1×10 8 cfu / mL, to obtain Pythium oligandrum liquid;

[0063] S3, diatomaceous earth and Bacillus subtilis liquid with a mass ratio of 1:1 are mixed, stirred uniformly, and then placed for 2h, and dried to obtain intermediate product 1;

[0064] Diatomaceous earth and Bacillus thuringiensis liquid with a mass ratio of 1:1 are mixed, stirred uniformly, and then placed for 2h, and dried to obtain intermediate product 2;

[0065] Diatomaceous earth and Paenibacillus polymyxa liquid with a mass ratio of 1:1 are mixed, stirred uniformly, and then placed for 2h, and dried to obtain intermediate product 3;

[0066] Diatomaceous earth and Pythium oligandrum liquid with a mass ratio of 1:1 are mixed, stirred uniformly, and then placed for 2h, and dried to obtain intermediate product 4;

[0067] S4, chitosan, phytanic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, dimethyl sulfoxide are blended in a mass ratio of 8:3:3:3:1000, stirred at 15℃ for 0.5h, heated to 65℃, stirred for 12h of reaction, cooled to 20℃, isopropanol (the mass ratio of dimethyl sulfoxide and isopropanol is 1:1) is added, stirred for 0.5h, filtered, washed and dried to obtain hydrophobic chitosan;

[0068] S5, hydrophobic chitosan and dimethyl sulfoxide are blended in a mass ratio of 1:48, heated to 40℃, stirred for 1h, cooled to room temperature, intermediate product 1 (the mass ratio of intermediate product 1 and hydrophobic chitosan is 8:3) is added and stirred for 1h, placed for 6h, washed and dried to obtain coated strain 1;

[0069] Hydrophobic chitosan and dimethyl sulfoxide are blended in a mass ratio of 1:48, heated to 40℃, stirred for 1h, cooled to room temperature, intermediate product 2 (the mass ratio of intermediate product 2 and hydrophobic chitosan is 8:3) is added and stirred for 1h, placed for 6h, washed and dried to obtain coated strain 2;

[0070] The hydrophobic chitosan and dimethyl sulfoxide were blended in a mass ratio of 1:48, heated to 40 DEG C, stirred for 1h, cooled to room temperature, and then the intermediate product 3 (the mass ratio of the intermediate product 3 to the hydrophobic chitosan was 8:3) was added and stirred for 1h, and then the mixture was allowed to stand for 6h, washed and dried to obtain the coated bacteria 3.

[0071] The hydrophobic chitosan and dimethyl sulfoxide were blended in a mass ratio of 1:48, heated to 40 DEG C, stirred for 1h, cooled to room temperature, and then the intermediate product 4 (the mass ratio of the intermediate product 4 to the hydrophobic chitosan was 8:3) was added and stirred for 1h, and then the mixture was allowed to stand for 6h, washed and dried to obtain the coated bacteria 4.

[0072] The coated bacteria 1, the coated bacteria 2, the coated bacteria 3 and the coated bacteria 4 were mixed in a mass ratio of 1:1:1:1 to obtain the salt-tolerant bacteria.

[0073] Example 3

[0074] A method for cultivating salt-tolerant bacteria, the method comprising the following steps:

[0075] S1, 3 parts of straw, 2 parts of mushroom cultivation waste, 0.2 parts of poultry manure, and 2 parts of inorganic salt were mixed, dried, crushed through a 100-mesh sieve, and then mixed with 10 parts of a culture medium base to obtain a biological waste-based culture medium;

[0076] S2, the activated Bacillus subtilis, Bacillus thuringiensis and Paenibacillus polymyxa were respectively added to the biological waste-based culture medium for expansion culture, and then the mixture was cultured at 30 DEG C for 48h, and then the concentration of the bacterial solution was adjusted to 1x10 8 cfu / mL to obtain a Bacillus subtilis bacterial solution, a Bacillus thuringiensis bacterial solution and a Paenibacillus polymyxa bacterial solution;

[0077] The activated Pythium oligandrum was added to the biological waste-based culture medium for expansion culture, and then the mixture was cultured at 26 DEG C for 72h, and then the concentration of the bacterial solution was adjusted to 1x10 8 cfu / mL to obtain a Pythium oligandrum bacterial solution;

[0078] S3, diatomite and the Bacillus subtilis bacterial solution were mixed in a mass ratio of 1:1, and then the mixture was stirred uniformly, allowed to stand for 2h, and then dried to obtain an intermediate product 1;

[0079] Diatomite and the Bacillus thuringiensis bacterial solution were mixed in a mass ratio of 1:1, and then the mixture was stirred uniformly, allowed to stand for 2h, and then dried to obtain an intermediate product 2;

[0080] Diatomite and the Paenibacillus polymyxa bacterial solution were mixed in a mass ratio of 1:1, and then the mixture was stirred uniformly, allowed to stand for 2h, and then dried to obtain an intermediate product 3;

[0081] Diatomite and the Pythium oligandrum bacterial solution were mixed in a mass ratio of 1:1, and then the mixture was stirred uniformly, allowed to stand for 2h, and then dried to obtain an intermediate product 4;

[0082] S4, chitosan, phytanic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, dimethyl sulfoxide were blended in a mass ratio of 8:2:3:3:1000, stirred at 15℃ for 0.5h, heated to 65℃, stirred for 12h, cooled to 20℃, isopropanol (the mass ratio of dimethyl sulfoxide and isopropanol was 1:1) was added, stirred for 0.5h, filtered, washed, and dried to obtain hydrophobic chitosan;

[0083] S5, hydrophobic chitosan and dimethyl sulfoxide were blended in a mass ratio of 1:48, heated to 40℃, stirred for 1h, cooled to room temperature, intermediate product 1 (the mass ratio of intermediate product 1 and hydrophobic chitosan was 8:3) was added and stirred for 1h, and stood for 6h, washed and dried to obtain coated strain 1;

[0084] hydrophobic chitosan and dimethyl sulfoxide were blended in a mass ratio of 1:48, heated to 40℃, stirred for 1h, cooled to room temperature, intermediate product 2 (the mass ratio of intermediate product 2 and hydrophobic chitosan was 8:3) was added and stirred for 1h, and stood for 6h, washed and dried to obtain coated strain 2;

[0085] hydrophobic chitosan and dimethyl sulfoxide were blended in a mass ratio of 1:48, heated to 40℃, stirred for 1h, cooled to room temperature, intermediate product 3 (the mass ratio of intermediate product 3 and hydrophobic chitosan was 8:3) was added and stirred for 1h, and stood for 6h, washed and dried to obtain coated strain 3;

[0086] hydrophobic chitosan and dimethyl sulfoxide were blended in a mass ratio of 1:48, heated to 40℃, stirred for 1h, cooled to room temperature, intermediate product 4 (the mass ratio of intermediate product 4 and hydrophobic chitosan was 8:3) was added and stirred for 1h, and stood for 6h, washed and dried to obtain coated strain 4;

[0087] coated strain 1, coated strain 2, coated strain 3, and coated strain 4 in a mass ratio of 1:1:1:1 were mixed to obtain salt-tolerant bacteria;

[0088] S6, 4 parts of salt-tolerant bacteria, 3 parts of urea, 5 parts of diammonium hydrogen phosphate, and 3 parts of potassium sulfate were mixed to obtain a product.

[0089] Comparative Example 1

[0090] Comparative Example 1 differs from Example 1 in that phytanic acid in step S4 is replaced by hexadecanoic acid, and the remaining components and preparation methods are the same as those of Example 1.

[0091] Comparative Example 2

[0092] Comparative Example 2 differs from Example 1 in that:

[0093] Step S4 is deleted, and step S5 is modified as follows: the intermediate product 1, the intermediate product 2, the intermediate product 3, and the intermediate product 4 in a mass ratio of 1:1:1:1 are mixed to obtain the salt-tolerant bacteria. The remaining components and preparation methods are the same as those in Example 1.

[0094] Comparative Example 3

[0095] Four parts of the salt-tolerant bacteria obtained in Comparative Example 1, three parts of urea, five parts of diammonium hydrogen phosphate, and three parts of potassium sulfate are mixed to obtain a product.

[0096] Comparative Example 4

[0097] Four parts of the salt-tolerant bacteria obtained in Comparative Example 2, three parts of urea, five parts of diammonium hydrogen phosphate, and three parts of potassium sulfate are mixed to obtain a product.

[0098] Test Example 1

[0099] The storage stability of Examples 1-3, Comparative Example 1, and Comparative Example 3 in a high-humidity environment is tested.

[0100] Test Method:

[0101] Examples 1-3, Comparative Example 1, and Comparative Example 3 are placed in a constant-temperature and constant-humidity environment at 37°C and a relative humidity of 80%, and are stored for 3 months, and the survival rate of the salt-tolerant bacteria is counted.

[0102] The test results are shown in Table 1.

[0103] Table 1 Test Results

[0104] Item Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 3 Salt-tolerant bacterial survival rate (%) 65 67 64 55 54

[0105] As can be seen from Table 1, the survival rate of the salt-tolerant bacteria of Examples 1-3 is significantly higher than that of Comparative Examples 1 and 3, because the hydrophobic chitosan of Examples 1-3 introduces phytanic acid containing multiple branched chains, which enhances the hydrophobicity of the salt-tolerant bacteria and avoids the adverse effects of high humidity on the salt-tolerant bacteria, while Comparative Examples 1 and 3 replace phytanic acid with hexadecanoic acid, which does not contain branched chains and cannot form a high-density branched carbon chain similar to the main chain and branched chain of phytanic acid, so its hydrophobicity is not as good as that of Examples 1-3, ultimately resulting in more significant adverse effects on the salt-tolerant bacteria in a high-humidity environment.

[0106] Test Example 2

[0107] The performance of the fertilizer samples prepared in Example 3 and Comparative Example 4 is tested.

[0108] Test Method:

[0109] 5 kg of local salinized soil (water content 15 wt%) was added to flowerpots, a total of 10 pots, divided into 2 groups, 5 pots each. Then 100 g / pot of the fertilizer sample prepared in Example 3 or Comparative Example 4 was added to each group, respectively, and mixed evenly.

[0110] After germination and seedling culture of the leaf lettuce seeds, seedlings with the same growth vigor (after the first true leaf emerged) were selected, transplanted into flowerpots at 3 plants / pot, and incubated at 25°C for 30 days. During this period, the management of the lettuce followed the agricultural operation.

[0111] The average weight of the aboveground part of the lettuce after culture was determined.

[0112] The test results are shown in Table 2.

[0113] Table 2 Test results

[0114] Item Example 3 Comparative Example 4 Lettuce weight / g 141.2 127.3

[0115] As can be seen from Table 2, the results of Example 3 are significantly better than those of Comparative Example 4, because Comparative Example 4 does not wrap the four intermediates with hydrophobic chitosan, but directly blends the four intermediates, so that the salt-tolerant bacteria on the four intermediates directly contact, cannot avoid the interaction of growth inhibition, etc., so that the synergistic effect of Example 3 cannot be formed, resulting in a decrease in effect.

[0116] It is apparent for those skilled in the art that the present application is not limited to the details of the foregoing exemplary embodiments, but can be implemented in other concrete forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be considered in all respects as illustrative and not restrictive, the scope of the present application being defined by the appended claims rather than the foregoing description, and it is intended that all changes falling within the meaning and range of equivalents of the claims be embraced therein.

[0117] In addition, it should be understood that although the present specification is described in terms of embodiments, each embodiment does not contain only one independent technical solution, and the description manner of the specification is only for the sake of clarity, those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be combined to form other embodiments that those skilled in the art can understand.

Claims

1. A method for breeding salt-tolerant bacteria, characterized by, The cultivation method of the salt-tolerant bacteria comprises the following steps: S1, mixing straw, mushroom cultivation waste, poultry manure, inorganic salt and culture medium matrix to obtain a biological waste-based culture medium; S2, culturing Bacillus subtilis, Bacillus thuringiensis and Paenibacillus polymyxa respectively using the biological waste-based culture medium to obtain a composite bacterial strain liquid; S3, mixing diatomite and the composite bacterial strain liquid, adsorbing and drying to obtain an intermediate product; S4, blending chitosan, phytanic acid and an activating agent, and reacting to obtain hydrophobic chitosan; S5, blending and stirring the intermediate product and the hydrophobic chitosan to obtain salt-tolerant bacteria; S6, mixing the salt-tolerant bacteria with nutrient substances; In step S3, the mass ratio of the diatomite to the composite bacterial strain liquid is 1:(1-3); The mass ratio of the chitosan, the phytanic acid and the activating agent is (6-10):(1-3):(5-7); In step S5, the mass ratio of the intermediate product to the hydrophobic chitosan is 8:

3.

2. The method for cultivating salt-tolerant bacteria according to claim 1, characterized in that, The concentration of the complex bacterial strain liquid in step S2 is 1-9x10 8 cfu / mL.

3. The method for cultivating salt-tolerant bacteria according to claim 1, characterized in that, In step S3, the mass ratio of the diatomite to the composite bacterial strain liquid is 1:(1-3).

4. The method for cultivating salt-tolerant bacteria according to claim 1, characterized in that, In step S4, the activating agent is selected from one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N-diisopropylcarbodiimide and N-hydroxysuccinimide.

5. The method for cultivating salt-tolerant bacteria according to claim 1, characterized in that, The nutrient substances comprise nitrogen fertilizer, phosphorus fertilizer and potassium fertilizer.