Cultivation method of halotolerant bacteria and application of halotolerant bacteria in agriculture

Abstract

The invention relates to a cultivation method of halotolerant bacteria and application of the halotolerant bacteria in agriculture, the cultivation method comprises the following steps: S1, mixing straw, mushroom cultivation waste, livestock manure, inorganic salt and a culture medium matrix to obtain a biological waste-based culture medium; s2, respectively culturing pythium aphanidermatum and bacillus by adopting the biological waste material-based culture medium to obtain a composite strain liquid; s3, diatomite and the composite strain liquid are mixed, adsorbed and dried, and an intermediate product is obtained; s4, blending chitosan, phytanic acid and an activating agent, and reacting to obtain hydrophobic chitosan; s5, blending and stirring the intermediate product and hydrophobic chitosan to obtain the halotolerant bacteria. The hydrophobic chitosan is beneficial to stable storage of the halotolerant bacteria in a high-humidity environment, and reduces adverse effects such as growth inhibition among different halotolerant bacteria, so that the different halotolerant bacteria can achieve a synergistic effect, and the efficacy of the fertilizer is finally improved.

Description

Technical Field

[0001] The present invention relates to the technical field of agriculture, and in particular to a method for cultivating salt-tolerant bacteria and its application in agriculture. Background Art

[0002] Saline-alkali land refers to land where excessive accumulation of salt (such as sodium chloride and sodium sulfate) or alkali (such as sodium carbonate and sodium bicarbonate) in the soil deteriorates soil properties and affects plant growth. Its core characteristics are excessive salt content in the surface or subsurface layers (typically exceeding 0.3%) or a significantly elevated pH (alkaline soils often have a pH greater than 9), which severely alters soil properties and makes it unsuitable for crop cultivation. The formation of saline-alkali land is caused by both natural and human factors. Natural factors include drought, intense evaporation, seawater intrusion, closed terrain, and salinity in the parent material. Human factors include inappropriate irrigation, improper fertilization and tillage, industry, and urbanization.

[0003] The hazards of saline-alkali land are mainly reflected in the multi-faceted negative impacts on soil, plants, ecosystems, and human production and life, including: 1. Soil function degradation: destruction of soil physical structure, deterioration of chemical properties, and inhibition of microbial activity; 2. Damage to agricultural production: crop production is restricted and reduced, and planting costs increase; 3. Ecosystem destruction: vegetation degradation and biodiversity decline, hydrological and climatic effects; 4. Economic and social impacts: threats to food security, high costs of ecological governance, and deterioration of the human living environment; 5. Long-term environmental risks: spread of secondary salinization and disruption of the ecological chain.

[0004] Improving and utilizing salinized land can improve low- and medium-yield fields and increase arable land area, which is of great significance for maintaining ecological and food security. Halophilic fertilizers are one of the core bioremediation technologies for agricultural production in saline-alkali land. Their significance lies not only in improving crop resistance and yield in the short term, but also in restoring the soil microbiome through the long-term effects of microorganisms, laying the foundation for the sustainable utilization of saline-alkali land. This technology, which combines scientific and economic feasibility, is a key breakthrough in alleviating the global salinized land crisis and ensuring food security.

[0005] However, there are still some problems with the use of fertilizers containing salt-tolerant bacteria, including: 1. During long-term storage, if the environmental humidity is high, the number of live bacteria may decrease, the activity may decrease, and the fertilizer effectiveness may decrease; 2. Different salt-tolerant bacteria may interact with each other, such as growth inhibition, resulting in a decrease in fertilizer effectiveness.

[0006] In summary, it is necessary to develop a new technical solution to overcome the defects in the existing technology. Summary of the Invention

[0007] The present invention provides a method for cultivating salt-tolerant bacteria and its application in agriculture. The culture medium for the salt-tolerant bacteria of the present invention uses agricultural waste materials such as straw, mushroom cultivation waste, and livestock manure, as well as inorganic salts, as raw materials. Pythium and Bacillus are separately cultivated to obtain a composite bacterial liquid. The liquid is then adsorbed on diatomaceous earth and then coated with hydrophobic chitosan. This method allows the salt-tolerant bacteria to be stably stored in high-humidity environments, reduces growth inhibition, and improves fertilizer effectiveness, thus having promising application prospects.

[0008] The object of the present invention is to provide a method for cultivating salt-tolerant bacteria, the method comprising the following steps:

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

[0010] S2. Using the biological waste-based culture medium to culture Pythium and Bacillus, respectively, to obtain a composite bacterial culture liquid;

[0011] S3, mixing the diatomaceous earth and the composite bacterial strain solution, adsorbing and drying to obtain an intermediate product;

[0012] S4, mixing chitosan, phytanic acid, and an activator to react to obtain hydrophobic chitosan;

[0013] S5. The intermediate product and hydrophobic chitosan are blended and stirred to obtain salt-tolerant bacteria.

[0014] Furthermore, it also includes:

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

[0016] Furthermore, in step S2, the Pythium is selected from Pythium oligandrum.

[0017] Furthermore, in step S2, the Bacillus is selected from one or more of Bacillus subtilis, Bacillus thuringiensis, and Paenibacillus polymyxa.

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

[0019] Furthermore, in step S2, the concentration of the composite bacterial culture liquid is 1-9×10 8 cfu / mL.

[0020] Furthermore, in step S3, the mass ratio of the diatomaceous earth to the composite bacterial culture liquid is 1:(1-3).

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

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

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

[0024] Furthermore, the nutrients include nitrogen fertilizer, phosphorus fertilizer and potassium fertilizer.

[0025] Furthermore, the nutrients include urea, diammonium hydrogen phosphate and potassium sulfate.

[0026] Furthermore, 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] Furthermore, the usage ratio of Pythium oligandrum, Bacillus subtilis, Bacillus thuringiensis and Paenibacillus polymyxa is (1-2):(1-2):(1-2):(1-2).

[0028] Another object of the present invention is to provide the application of the above-mentioned method for cultivating salt-tolerant bacteria in agriculture.

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

[0030] The present invention provides a method for cultivating salt-tolerant bacteria. The method uses agricultural waste materials such as straw, mushroom cultivation waste, and livestock manure, and inorganic salts as raw materials to cultivate Pythium oligandrum, Bacillus subtilis, Bacillus thuringiensis, and Bacillus polymyxa. A composite bacterial strain liquid is obtained, which is adsorbed with diatomaceous earth and then wrapped with hydrophobic chitosan to obtain salt-tolerant bacteria. The salt-tolerant bacteria are mixed with nutrients to obtain fertilizer. The hydrophobic chitosan is obtained by activating the carboxyl group on phytanic acid with an activator and reacting it with the amino group of chitosan, thereby introducing a multi-branched alkyl acid on the chitosan. The hydrophobic chitosan is then blended with the intermediate product to produce hydrogen bonds and other interactions, so that the hydrophobic chitosan is wrapped around the surface of the intermediate product to form a protective layer. The above-mentioned multi-branched alkyl acid makes chitosan hydrophobic, which is beneficial for the stable storage of salt-tolerant bacteria in a high humidity environment. The protective layer formed by 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, ultimately improving the effectiveness of the fertilizer.

[0031] Diatomaceous earth contains rich pore structure and has good adsorption effect. It can be used as a carrier of salt-tolerant bacteria to fix salt-tolerant bacteria, which is beneficial to the preservation and growth of salt-tolerant bacteria.

[0032] Agricultural waste materials such as straw, mushroom cultivation waste, and livestock manure used in the present invention are rich in nutrients such as cellulose, starch, protein, and amino acids. Cultivating salt-tolerant bacteria using these agricultural waste materials as raw materials not only realizes the utilization of waste materials but also avoids the pollution of the waste materials to the environment, which is beneficial to the sustainable development of agriculture. In addition, the added inorganic salts can simulate the salt stress environment, and by adjusting the osmotic pressure, screening salt-tolerant strains, maintaining cell functions, and inducing the secretion of metabolites, ensure that the salt-tolerant bacteria maintain their salt-tolerant characteristics and functional activity during the cultivation process, ultimately ensuring their application effect in saline-alkali environments. DETAILED DESCRIPTION

[0033] In order to more clearly illustrate the technical solutions of the present invention, the following examples are given. Unless otherwise stated, the raw materials, reactions and post-processing methods mentioned in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0034] The terms "preferred," "preferably," "more preferred," and the like, used herein, refer to embodiments of the invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0035] It should be understood that, except in any operating examples, or where otherwise indicated, all numbers expressing, for example, quantities of ingredients used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximations that may vary depending upon the desired properties to be obtained by the present invention.

[0036] In the examples of the present invention, Bacillus subtilis (GDMCC NO.1.131), Bacillus thuringiensis (GDMCC NO.1.126), and Paenibacillus polymyxa (GDMCC NO.1.1630) were purchased from Guangdong Provincial Microbial Culture Collection Center, and Pythium oligandrum (GSICC 61615) was purchased from Gansu Provincial Industrial Microbial Culture Collection Center.

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

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

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

[0040] The activators in the embodiment of the present invention are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide.

[0041] The inorganic salts in the embodiment of the present invention are NaCl and KCl in a mass ratio of 1:1.

[0042] The “parts” in the embodiments of the present invention refer to parts by mass.

[0043] Example 1

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

[0045] S1. Mix 3 parts of straw, 2 parts of mushroom cultivation waste, 0.2 parts of livestock manure, and 2 parts of inorganic salts, dry them, grind them through a 100-mesh sieve, and mix them with 10 parts of culture medium to obtain a biowaste-based culture medium;

[0046] S2, adding the activated Bacillus subtilis, Bacillus thuringiensis and Paenibacillus polymyxa to the biological waste-based culture medium for expansion culture, culturing at 30°C for 48h, and adjusting the bacterial solution concentration to 1×10 8 cfu / mL, and obtained Bacillus subtilis, Bacillus thuringiensis, and Paenibacillus polymyxa bacterial solutions;

[0047] The activated Pythium oligandrum was added to the biological waste-based culture medium for expansion culture, cultured at 26°C for 72 hours, and the bacterial solution concentration was adjusted to 1×10 8 cfu / mL, and the Pythium oligandrum bacterial solution was obtained;

[0048] S3, mixing diatomaceous earth and Bacillus subtilis liquid in a mass ratio of 1:1, stirring evenly, letting it stand for 2 hours, and drying to obtain intermediate product 1;

[0049] Mix diatomaceous earth and Bacillus thuringiensis liquid in a mass ratio of 1:1, stir evenly, let stand for 2 hours, and dry to obtain intermediate product 2;

[0050] Mix diatomaceous earth and Paenibacillus polymyxa bacterial solution in a mass ratio of 1:1, stir evenly, let stand for 2 hours, and dry to obtain intermediate product 3;

[0051] Mix diatomaceous earth and Pythium oligandrum liquid in a mass ratio of 1:1, stir evenly, let stand for 2 hours, and dry to obtain intermediate product 4;

[0052] S4, chitosan, phytanic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and dimethyl sulfoxide were blended in a mass ratio of 8:2:3:3:1000, stirred at 15°C for 0.5h, heated to 65°C, stirred for 12h, cooled to 20°C, and isopropyl alcohol (the mass ratio of dimethyl sulfoxide to isopropyl alcohol was 1:1) was added, stirred for 0.5h, filtered, washed, and dried to obtain hydrophobic chitosan;

[0053] S5. Blend hydrophobic chitosan and dimethyl sulfoxide in a mass ratio of 1:48, heat to 40° C., stir for 1 h, cool to room temperature, add intermediate 1 (the mass ratio of intermediate 1 to hydrophobic chitosan is 8:3), stir for 1 h, let stand for 6 h, wash, and dry to obtain coated strain 1;

[0054] Hydrophobic chitosan and dimethyl sulfoxide were blended at a mass ratio of 1:48, heated to 40°C, stirred for 1 hour, cooled to room temperature, added with intermediate product 2 (the mass ratio of intermediate product 2 to hydrophobic chitosan was 8:3), stirred for 1 hour, allowed to stand for 6 hours, washed, and dried to obtain coated strain 2;

[0055] Hydrophobic chitosan and dimethyl sulfoxide were blended at a mass ratio of 1:48, heated to 40°C, stirred for 1 hour, cooled to room temperature, added with intermediate product 3 (the mass ratio of intermediate product 3 to hydrophobic chitosan was 8:3), stirred for 1 hour, allowed to stand for 6 hours, washed, and dried to obtain coated strain 3;

[0056] Hydrophobic chitosan and dimethyl sulfoxide were blended at a mass ratio of 1:48, heated to 40°C, stirred for 1 hour, cooled to room temperature, added with intermediate product 4 (the mass ratio of intermediate product 4 to hydrophobic chitosan was 8:3), stirred for 1 hour, allowed to stand for 6 hours, washed, and dried to obtain coated strain 4;

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

[0058] Example 2

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

[0060] S1. Mix 3 parts of straw, 2 parts of mushroom cultivation waste, 0.2 parts of livestock manure, and 2 parts of inorganic salts, dry them, grind them through a 100-mesh sieve, and mix them with 10 parts of culture medium to obtain a biowaste-based culture medium;

[0061] S2, adding the activated Bacillus subtilis, Bacillus thuringiensis and Paenibacillus polymyxa to the biological waste-based culture medium for expansion culture, culturing at 30°C for 48h, and adjusting the bacterial solution concentration to 1×10 8 cfu / mL, and obtained Bacillus subtilis, Bacillus thuringiensis, and Paenibacillus polymyxa bacterial solutions;

[0062] The activated Pythium oligandrum was added to the biological waste-based culture medium for expansion culture, cultured at 26°C for 72 hours, and the bacterial solution concentration was adjusted to 1×10 8 cfu / mL, and the Pythium oligandrum bacterial solution was obtained;

[0063] S3, mixing diatomaceous earth and Bacillus subtilis liquid in a mass ratio of 1:1, stirring evenly, letting it stand for 2 hours, and drying to obtain intermediate product 1;

[0064] Mix diatomaceous earth and Bacillus thuringiensis liquid in a mass ratio of 1:1, stir evenly, let stand for 2 hours, and dry to obtain intermediate product 2;

[0065] Mix diatomaceous earth and Paenibacillus polymyxa bacterial solution in a mass ratio of 1:1, stir evenly, let stand for 2 hours, and dry to obtain intermediate product 3;

[0066] Mix diatomaceous earth and Pythium oligandrum liquid in a mass ratio of 1:1, stir evenly, let stand for 2 hours, and dry to obtain intermediate product 4;

[0067] S4, blending chitosan, phytanic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and dimethyl sulfoxide in a mass ratio of 8:3:3:3:1000, stirring at 15°C for 0.5h, heating to 65°C, stirring for 12h, cooling to 20°C, adding isopropyl alcohol (the mass ratio of dimethyl sulfoxide to isopropyl alcohol is 1:1), stirring for 0.5h, filtering, washing, and drying to obtain hydrophobic chitosan;

[0068] S5. Blend hydrophobic chitosan and dimethyl sulfoxide in a mass ratio of 1:48, heat to 40° C., stir for 1 h, cool to room temperature, add intermediate 1 (the mass ratio of intermediate 1 to hydrophobic chitosan is 8:3), stir for 1 h, let stand for 6 h, wash, and dry to obtain coated strain 1;

[0069] Hydrophobic chitosan and dimethyl sulfoxide were blended at a mass ratio of 1:48, heated to 40°C, stirred for 1 hour, cooled to room temperature, added with intermediate product 2 (the mass ratio of intermediate product 2 to hydrophobic chitosan was 8:3), stirred for 1 hour, allowed to stand for 6 hours, washed, and dried to obtain coated strain 2;

[0070] Hydrophobic chitosan and dimethyl sulfoxide were blended at a mass ratio of 1:48, heated to 40°C, stirred for 1 hour, cooled to room temperature, added with intermediate product 3 (the mass ratio of intermediate product 3 to hydrophobic chitosan was 8:3), stirred for 1 hour, allowed to stand for 6 hours, washed, and dried to obtain coated strain 3;

[0071] Hydrophobic chitosan and dimethyl sulfoxide were blended at a mass ratio of 1:48, heated to 40°C, stirred for 1 hour, cooled to room temperature, added with intermediate product 4 (the mass ratio of intermediate product 4 to hydrophobic chitosan was 8:3), stirred for 1 hour, allowed to stand for 6 hours, washed, and dried to obtain coated strain 4;

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

[0073] Example 3

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

[0075] S1. Mix 3 parts of straw, 2 parts of mushroom cultivation waste, 0.2 parts of livestock manure, and 2 parts of inorganic salts, dry them, grind them through a 100-mesh sieve, and mix them with 10 parts of culture medium to obtain a biowaste-based culture medium;

[0076] S2, adding the activated Bacillus subtilis, Bacillus thuringiensis and Paenibacillus polymyxa to the biological waste-based culture medium for expansion culture, culturing at 30°C for 48h, and adjusting the bacterial solution concentration to 1×10 8 cfu / mL, and obtained Bacillus subtilis, Bacillus thuringiensis, and Paenibacillus polymyxa bacterial solutions;

[0077] The activated Pythium oligandrum was added to the biological waste-based culture medium for expansion culture, cultured at 26°C for 72 hours, and the bacterial solution concentration was adjusted to 1×10 8 cfu / mL, and the Pythium oligandrum bacterial solution was obtained;

[0078] S3, mixing diatomaceous earth and Bacillus subtilis liquid in a mass ratio of 1:1, stirring evenly, letting it stand for 2 hours, and drying to obtain intermediate product 1;

[0079] Mix diatomaceous earth and Bacillus thuringiensis liquid in a mass ratio of 1:1, stir evenly, let stand for 2 hours, and dry to obtain intermediate product 2;

[0080] Mix diatomaceous earth and Paenibacillus polymyxa bacterial solution in a mass ratio of 1:1, stir evenly, let stand for 2 hours, and dry to obtain intermediate product 3;

[0081] Mix diatomaceous earth and Pythium oligandrum liquid in a mass ratio of 1:1, stir evenly, let stand for 2 hours, and dry to obtain intermediate product 4;

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

[0083] S5. Blend hydrophobic chitosan and dimethyl sulfoxide in a mass ratio of 1:48, heat to 40° C., stir for 1 h, cool to room temperature, add intermediate 1 (the mass ratio of intermediate 1 to hydrophobic chitosan is 8:3), stir for 1 h, let stand for 6 h, wash, and dry to obtain coated strain 1;

[0084] Hydrophobic chitosan and dimethyl sulfoxide were blended at a mass ratio of 1:48, heated to 40°C, stirred for 1 hour, cooled to room temperature, added with intermediate product 2 (the mass ratio of intermediate product 2 to hydrophobic chitosan was 8:3), stirred for 1 hour, allowed to stand for 6 hours, washed, and dried to obtain coated strain 2;

[0085] Hydrophobic chitosan and dimethyl sulfoxide were blended at a mass ratio of 1:48, heated to 40°C, stirred for 1 hour, cooled to room temperature, added with intermediate product 3 (the mass ratio of intermediate product 3 to hydrophobic chitosan was 8:3), stirred for 1 hour, allowed to stand for 6 hours, washed, and dried to obtain coated strain 3;

[0086] Hydrophobic chitosan and dimethyl sulfoxide were blended at a mass ratio of 1:48, heated to 40°C, stirred for 1 hour, cooled to room temperature, added with intermediate product 4 (the mass ratio of intermediate product 4 to hydrophobic chitosan was 8:3), stirred for 1 hour, allowed to stand for 6 hours, washed, and dried to obtain coated strain 4;

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

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

[0089] Comparative Example 1

[0090] The difference between Comparative Example 1 and Example 1 is that the phytanic acid in step S4 is replaced by hexadecanoic acid, and the remaining components and preparation method are the same as those in Example 1.

[0091] Comparative Example 2

[0092] The difference between Comparative Example 2 and Example 1 is that:

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

[0094] Comparative Example 3

[0095] 4 parts of the salt-tolerant bacteria obtained in Comparative Example 1, 3 parts of urea, 5 parts of diammonium hydrogen phosphate and 3 parts of potassium sulfate were mixed to obtain a product.

[0096] Comparative Example 4

[0097] 4 parts of the salt-tolerant bacteria obtained in Comparative Example 2, 3 parts of urea, 5 parts of diammonium hydrogen phosphate and 3 parts of potassium sulfate were mixed to obtain a product.

[0098] Test Example 1

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

[0100] Test method:

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

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

[0103] Table 1 Test results

[0104] project Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 3 Survival rate of salt-tolerant bacteria (%) 65 67 64 55 54

[0105] As can be seen from Table 1, the survival rate of the salt-tolerant bacteria in Examples 1-3 is significantly higher than that in Comparative Example 1 and Comparative Example 3. This is because the hydrophobic chitosan in Examples 1-3 introduces a multi-branched alkyl acid (phytanic acid), which enhances the hydrophobicity of the salt-tolerant bacteria and avoids the adverse effects of a high humidity environment on the salt-tolerant bacteria. In Comparative Examples 1 and 3, phytanic acid is replaced with hexadecanoic acid, which does not contain branches and cannot form a main chain similar to that of phytanic acid and a high-density branched carbon chain with crossed branches, making its hydrophobicity inferior to that of Examples 1-3, which ultimately leads to a more obvious adverse effect on the salt-tolerant bacteria in a high humidity environment.

[0106] Test Example 2

[0107] The performance tests were performed on the fertilizer samples prepared in Example 3 and Comparative Example 4.

[0108] Test method:

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

[0110] After germinating the variegated lettuce seeds and raising them into seedlings, seedlings with the same growth potential (after growing the first true leaf) were selected and transplanted into flower pots at a rate of 3 plants per pot. The plants were cultured at a constant temperature of 25°C for 30 days. During this period, the management of the lettuce followed the agricultural season operation.

[0111] The average weight of the above-ground part of the lettuce after cultivation was measured.

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

[0113] Table 2 Test results

[0114] project 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. This is because in Comparative Example 4, the four intermediate products are not wrapped with hydrophobic chitosan, but the four intermediate products are directly blended, so that the salt-tolerant bacteria on the four intermediate products are in direct contact, and interactions such as growth inhibition cannot be avoided, thereby failing to form the synergistic effect of Example 3, resulting in a decrease in the effect.

[0116] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0117] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for cultivating salt-tolerant bacteria, characterized in that: The method for cultivating the salt-tolerant bacteria comprises the following steps: S1, mixing straw, mushroom cultivation waste, livestock manure, inorganic salts and a culture medium matrix to obtain a biological waste-based culture medium; S2. Using the biological waste-based culture medium to culture Pythium and Bacillus, respectively, to obtain a composite bacterial culture liquid; S3, mixing the diatomaceous earth and the composite bacterial strain solution, adsorbing and drying to obtain an intermediate product; S4, mixing chitosan, phytanic acid, and an activator to react to obtain hydrophobic chitosan; S5. The intermediate product and hydrophobic chitosan are blended and stirred to obtain salt-tolerant bacteria.

2. The method for cultivating salt-tolerant bacteria according to claim 1, wherein Also includes: S6. Mix the salt-tolerant bacteria with nutrients.

3. The method for cultivating salt-tolerant bacteria according to claim 1, wherein In step S2, the concentration of the composite bacterial culture solution is 1-9×10 8 cfu / mL.

4. The method for cultivating salt-tolerant bacteria according to claim 1, wherein In step S3, the mass ratio of the diatomaceous earth to the composite bacterial culture liquid is 1:(1-3).

5. The method for cultivating salt-tolerant bacteria according to claim 1, wherein In step S4, the mass ratio of chitosan, phytanic acid and activator is (6-10):(1-3):(5-7).

6. The method for cultivating salt-tolerant bacteria according to claim 1, wherein 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.

7. The method for cultivating salt-tolerant bacteria according to claim 1, wherein In step S5, the mass ratio of the intermediate product to the hydrophobic chitosan is 8:(1-3).

8. The method for cultivating salt-tolerant bacteria according to claim 2, wherein: The nutrients include nitrogen fertilizer, phosphorus fertilizer and potash fertilizer.

9. Application of the method for cultivating salt-tolerant bacteria according to any one of claims 1 to 8 in agriculture.